Novel human genes relating to respiratory diseases and obesity

ABSTRACT

This invention relates to isolated nucleic acids comprising genes of human chromosome 12q23-qter and the proteins encoded by these genes. Expression vectors and host cells containing such genes or fragments thereof, as well as antibodies to the proteins encoded by these nucleic acids are also included herein.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 12/180,184, to be issued as U.S. Pat. No. 7,928,200, filed onJul. 25, 2008; which is a divisional of U.S. patent application Ser. No.10/743,704, now U.S. Pat. No. 7,407,804, filed on Dec. 22, 2003, whichis a divisional application of U.S. patent application Ser. No.09/627,465, now U.S. Pat. No. 6,737,519, filed on Jul. 28, 2000, whichclaims the benefit of U.S. Provisional Application Ser. No. 60/146,336filed Jul. 30, 1999 and U.S. Provisional Application Ser. No. 60/211,749filed Jun. 14, 2000, the entire teachings of all are incorporated hereinby reference.

FIELD OF THE INVENTION

The present invention relates generally to isolated nucleic acids andthe classification of the same. The invention more particularly relatesto a novel gene and novel nucleic acids related to asthma and otherrespiratory diseases and the classification and therapeutic anddiagnostic uses of this gene.

BACKGROUND

Wilkinson et al. showed linkage of asthma to markers on human chromosome12 (Genomics, 53: 251-259 (1998)). In addition, Wilson et al. has shownthat obesity may be linked to asthma (Arch. Intern. Med. 159: 2513-14(1999)). In particular chromosomal region 12q23-qter has been linked toa variety of genetic disorders including male germ cell tumors,histidinemia, growth retardation with deafness and mental retardation,deficiency of Acyl-CoA dehydrogenase, spinal muscular atrophy, Darierdisease, cardiomyopathy, Spinocerebellar ataxia-2, brachydactyl),Mevalonicaciduria, Hyperimmunoglobulinemia D, Noonan syndrome-1,Cardiofaciocutaneous syndrome, spinal muscular atrophy-4, tyrosinemia,phenylketonuria, B-cell non-Hodgkin lymphoma, Ulnar-mammary syndrome,Holt-Oram syndrome, Scapuloperoneal spinal muscular atrophy, alcoholintolerance, MODY, Diabetes mellitus, noninsulin-dependent, 2 anddiabetes mellitus insulin-dependent (See National Center forBiotechnology Information: at the website of: (hypertext transferprotocol, (i.e., http), world wide web (i.e., www), National Center forBiotechnology Information (ncbi).National Library of Medicine(nlm).National Institutes of Health (NIH).Government (gov)/omim.).Although this region appears to contain genes affecting these disordersfew genes have been discovered. There is a need in the art foridentifying specific genes for such disorders because they are alsoassociated with obesity and lung disease, particularly inflammatory lungdisease phenotypes such as Chronic Obstructive Lung Disease (COPD),Adult Respiratory Distress Syndrome (ARDS), and asthma. Identificationand characterization of such genetic compositions will make possible thedevelopment of effective diagnostics and therapeutic means to treat lungrelated disorders as well as the other diseases described herein.

SUMMARY OF THE INVENTION

This invention relates to Gene 214 located on chromosome 12q23-qter.Nucleic acids comprising all or a part of, or complementary fragments ofGene 214 and cDNA are described in various embodiments. Vectors and hostcells containing the nucleic acids herein described are also included inthis invention. These nucleic acids can be used in therapeuticapplications for a multitude of diseases either through theoverexpression of a recombinant nucleic acid comprising all or a portionof a Gene 214 gene, or by the use of these oligonucleotides and genes tomodulate the expression of an endogenous gene or the activity of anendogenous gene product. Examples of therapeutic approaches includeanti-sense inhibition of gene expression, gene therapy, monoclonalantibodies that specifically bind to the gene products, and the like. Invitro expression of the recombinant gene products can also be obtained.

Diagnostic methods are also described which utilize all or part of thenucleic acids of this invention. Such nucleic acids can be used, forexample, as part of diagnostic methods to identify Gene 214 nucleicacids to screen for a predisposition to various genetic diseases. Inaddition, nucleic acids described herein can be used to identifychromosomal abnormalities within the chromosomal region 12q23-qter.

Further, this invention identifies various single nucleotidepolymorphisms (SNPs) within several of the nucleic acids describedherein. Some of these polymorphisms also comprise changes to thepolypeptides of the present invention. The SNPs, together with thewild-type alleles can be used to prepare specific probes for detectionof various disease states in an individual. Thus, in one embodiment,this invention provides a method of detecting chromosome abnormalitieson chromosome 12q23-qter.

Proteins, polypeptides, and peptides encoded by all or a part of thenucleic acids comprising Gene 214 are included in this invention. Suchamino acid sequences are useful for diagnostic and therapeutic purposes.Further, antibodies can be raised against all or a part of these aminoacid sequences for specific diagnostic and therapeutic methods requiringsuch antibodies. These antibodies can be polyclonal, monoclonal, orantibody fragments.

In a further embodiment, vectors and host cells containing vectors whichcomprise all or a portion of the nucleic acid sequences of thisinvention can be constructed for nucleic acid preparations, includinganti-sense, and/or for expression of encoded proteins and polypeptides.Such host cells can be prokaryotic or eukaryotic cells.

Still another embodiment of the invention comprises a method ofidentifying a protein which is a candidate for being involved in asthma(a “candidate protein”). Candidate proteins are identified by a processcomprising (i) identifying a protein in a first individual having theasthma phenotype; (ii) identifying a protein in a second individual nothaving the asthma phenotype; comparing the protein of the firstindividual to the protein of the second individual, wherein (a) theprotein that is present in the second individual but not the firstindividual is the candidate protein or (b) the protein that is presentin a higher amount in the second individual than in the first individualis the candidate protein or (c) the protein that is present in a loweramount in the second individual than in the first individual is thecandidate protein.

This invention also includes nonhuman transgenic animals containing oneor more of the nucleic acids of this invention for screening and otherpurposes. Further, knockout nonhuman transgenic animals can be producedwherein one or more endogenous genes or portions of such genescorresponding to the nucleic acids of this invention are replaced bymarker genes or are deleted.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the plot of multipoint LOD score against the map locationof the markers along chromosome 12.

FIG. 2 depicts the STS content of the 12q23-qter BAC RP11-0702C13containing Gene 214

FIGS. 3A-3C depict the nucleotide (SEQ ID NO: 2) and amino acid sequence(SEQ ID NO: 3) of Gene 214a.

FIGS. 4A-4C depict the nucleotide (SEQ ID NO: 4) and amino acid sequence(SEQ ID NO: 5) of Gene 214b.

FIGS. 5A-5C depict the nucleotide (SEQ ID NO: 6) and amino acid sequence(SEQ ID NO: 7) of Gene 214c.

FIGS. 6A-6D depict the nucleotide (SEQ ID NO: 8) and amino acid sequence(SEQ ID NO: 9) of Gene 214d.

FIGS. 7A-7D depict the nucleotide (SEQ ID NO: 10) and amino acidsequence (SEQ ID NO: 11) of Gene 214e.

FIGS. 8A-8B show a schematic view of the exons of Gene 214a, 214b, 214c,214d, and 214e and the corresponding single nucleotide polymorphisms.

FIG. 9 shows a Northern Analysis of Gene 214.

FIG. 10A-10B depict the nucleic acid sequence of the exons of Gene 214:Exon A—SEQ ID NO: 38; Exon B—SEQ ID NO: 39; Exon C—SEQ ID NO: 40; ExonC.2—SEQ ID NO: 41; Exon E.1—SEQ ID NO: 42; Exon E.2—SEQ ID NO: 43; ExonE.3—SEQ ID NO: 44; Exon F—SEQ ID NO: 45.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to Gene 214 nucleic acids comprisinggenomic DNA within BAC RP11-0702C13, the corresponding cDNA sequences,RNA, fragments of the genomic, cDNA, or RNA nucleic acids comprising 20,40, 60, 100, 200, 500 or more contiguous nucleotides, and thecomplements thereof. Closely related variants are also included as partof this invention, as well as recombinant nucleic acids comprising atleast 50, 60, 70, 80, or 90% of the nucleic acids described above whichwould be identical to a Gene 214 nucleic acids except for one or a fewsubstitutions, deletions, or additions.

Further, the nucleic acids of this invention include the adjacentchromosomal regions of Gene 214 required for accurate expression of therespective gene. In a preferred embodiment, the present invention isdirected to at least 15 contiguous nucleotides of the nucleic acidsequence of any of SEQ ID NO:2 (FIGS. 3A-3C), SEQ ID NO:4 (FIGS. 4A-4C),SEQ ID NO:6 (FIGS. 5A-5C), SEQ ID NO: 8 (FIGS. 6A-6D), and SEQ ID NO:10(FIGS. 7A-7D). More particularly, embodiments of this invention includethe BAC clone containing segments of Gene 214 including RP11-0702C13. Apreferred embodiment is the nucleotide sequence of the BAC clonesconsisting of SEQ ID NO:1.

This invention further relates to methods using isolated and/orrecombinant nucleic acids (DNA or RNA) that are characterized by theirability to hybridize to (a) a nucleic acid encoding a protein orpolypeptide, such as a nucleic acid having any of the sequences of SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, and SEQ ID NO:10 or (b)a portion of the foregoing (e.g., a portion comprising the minimumnucleotides of the Gene 214 nucleic acid code a functional Gene 214protein or the minimum number to inhibit an endogenous Gene 214; or bytheir ability to encode a polypeptide having the amino acid sequence ofSEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 9 and SEQ ID NO: 11 orto encode functional equivalents thereof; e.g., a polypeptide which whenincorporated into a cell, has all or part of the activity of a Gene 214protein, or by both characteristics. A functional equivalent of a Gene214 protein, therefore, would have a similar amino acid sequence (atleast 65% sequence identity) and similar characteristics to, or performin substantially the same way as Gene 214 protein. A nucleic acid whichhybridizes to a nucleic acid encoding a Gene 214 protein or polypeptide,such as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, and SEQ IDNO:10 can be double- or single-stranded. Hybridization to DNA such asDNA having the sequence SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO: 8, and SEQ ID NO:10 includes hybridization to the strand shown orits complementary strand.

In one embodiment, the percent amino acid sequence similarity between aGene 214 polypeptide such as SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQID NO: 9 and SEQ ID NO: 11, and functional equivalents thereof is atleast about 50%. In a preferred embodiment, the percent amino acidsequence similarity between such a Gene 214 polypeptide and itsfunctional equivalents is at least about 65%. More preferably, thepercent amino acid sequence similarity between a Gene 214 polypeptideand its functional equivalents is at least about 75%, and still morepreferably, at least about 80%. To determine percent nucleotide or aminoacid sequence similarity, sequences can be compared to publiclyavailable sequence databases (National Center for BiotechnologyInformation, National Library of Medicine, 38A, 8N905, 8600 RockvillePike, Bethesda, Md. 20894; www.ncbi.nlm.nih.gov) using the blastn2algorithm (Altsch, Nucl. Acids Res., 25:3389-3402 (1997)). Theparameters for a typical search are: E=0.05, v=50, B=50 (where E is theexpected probability score cutoff, V is the number of database entriesreturned in the reporting of the results, and B is the number ofsequence alignments returned in the reporting of the results (Altsch etal, J. Mol. Biol., 215:403-410 (1990)).

Isolated and/or recombinant nucleic acids meeting these criteriacomprise nucleic acids having sequences identical to sequences ofnaturally occurring Gene 214 genes such as Gene 214a, Gene 214b, Gene214c, Gene 214d, Gene 214e, and portions thereof, or variants of thenaturally occurring genes. Such variants include mutants differing bythe addition, deletion or substitution of one or more nucleotides,modified nucleic acids in which one or more nucleotides are modified(e.g., DNA or RNA analogs), and mutants comprising one or more modifiednucleotides including repeated fragments.

Such nucleic acids, including DNA or RNA, can be detected and isolatedby hybridization under high stringency conditions or moderate stringencyconditions, for example, which are chosen so as to not permit thehybridization of nucleic acids having non-complementary sequences.“Stringency conditions” for hybridizations is a term of art which refersto the conditions of temperature and buffer concentration which permithybridization of a particular nucleic acid to another nucleic acid inwhich the first nucleic acid may be perfectly complementary to thesecond, or the first and second may share some degree of complementaritywhich is less than perfect. For example, certain high stringencyconditions can be used which distinguish perfectly complementary nucleicacids from those of less complementarity. “High stringency conditions”and “moderate stringency conditions” for nucleic acid hybridizations areexplained on pages 2.10.1-2.10.16 (see particularly 2.10.8-11) and pages6.3.1-6 in Current Protocols in Molecular Biology (Ausubel, F. M. etal., eds., Vol. 1, containing supplements up through Supplement 29,1995), the teachings of which are hereby incorporated by reference. Theexact conditions which determine the stringency of hybridization dependnot only on ionic strength, temperature and the concentration ofdestabilizing agents such as formamide, but also on factors such as thelength of the nucleic acid sequence, base composition, percent mismatchbetween hybridizing sequences and the frequency of occurrence of subsetsof that sequence within other non-identical sequences. Thus, high ormoderate stringency conditions can be determined empirically.

High stringency hybridization procedures (1) employ low ionic strengthand high temperature for washing, such as 0.015 M NaCl/0.0015 M sodiumcitrate, pH 7.0 (0.1×SSC) with 0.1% sodium dodecyl sulfate (SDS) at 50°C.; (2) employ during hybridization 50% (vol/vol) formamide with5×Denhardt's solution (0.1% weight/volume highly purified bovine serumalbumin/0.1% wt/vol Ficoll/0.1% wt/vol polyvinylpyrrolidone), 50 mMsodium phosphate buffer at pH 6.5 and 5×SSC at 42° C.; or (3) employhybridization with 50% formamide, 5×SSC, 50 mM sodium phosphate (pH6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmonsperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., withwashes at 42° C. in 0.2×SSC and 0.1% SDS.

By varying hybridization conditions from a level of stringency at whichno hybridization occurs to a level at which hybridization is firstobserved, conditions which will allow a given sequence to hybridize withthe most similar sequences in the sample can be determined. Preferablythe hybridizing sequences will have 60-70% sequence identity, morepreferably 70-85% sequence identity, and even more preferably 90-100%sequence identity.

Exemplary conditions are described in Krause, M. H. and S. A. Aaronson(1991) Methods in Enzymology, 200:546-556. Also, see especially page2.10.11 in Current Protocols in Molecular Biology (supra), whichdescribes how to determine washing conditions for moderate or lowstringency conditions. Washing is the step in which conditions areusually set so as to determine a minimum level of complementarity of thehybrids. Generally, from the lowest temperature at which only homologoushybridization occurs, a 1% mismatch between hybridizing nucleic acidsresults in a 1° C. decrease in the melting temperature T_(m), for anychosen SSC concentration. Generally, doubling the concentration of SSCresults in an increase in T_(m) of ˜17° C. Using these guidelines, thewashing temperature can be determined empirically for moderate or lowstringency, depending on the level of mismatch sought.

Isolated and/or recombinant nucleic acids that are characterized bytheir ability to hybridize to (a) a nucleic acid encoding a Gene 214polypeptide, such as the nucleic acids depicted as SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO: 8, and SEQ ID NO:10, b) the complement,(c) or a portion of (a) or (b) (e.g. under high or moderate stringencyconditions), may further encode a protein or polypeptide having at leastone function characteristic of a Gene 214 polypeptide, such asprotective barrier of the respiratory epithelium activity, or binding ofantibodies that also bind to non-recombinant Gene 214 protein orpolypeptide. The catalytic or binding function of a protein orpolypeptide encoded by the hybridizing nucleic acid may be detected bystandard enzymatic assays for activity or binding (e.g., assays whichmeasure the binding of a transit peptide or a precursor, or othercomponents of the translocation machinery). Enzymatic assays,complementation tests, or other suitable methods can also be used inprocedures for the identification and/or isolation of nucleic acidswhich encode a polypeptide such as a polypeptide of the amino acidsequences SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 9 and SEQ IDNO: 11, or a functional equivalent of these polypeptides. The antigenicproperties of proteins or polypeptides encoded by hybridizing nucleicacids can be determined by immunological methods employing antibodiesthat bind to a Gene 214 polypeptide such as immunoblot,immunoprecipitation and radioimmunoassay. PCR methodology, includingRAGE (Rapid Amplification of Genomic DNA Ends), can also be used toscreen for and detect the presence of nucleic acids which encode Gene214-like proteins and polypeptides, and to assist in cloning suchnucleic acids from genomic DNA. PCR methods for these purposes can befound in Innis, M. A., et al. (1990) PCR Protocols: A Guide to Methodsand Applications, Academic Press, Inc., San Diego, Calif., incorporatedherein by reference.

It is understood that, as a result of the degeneracy of the geneticcode, many nucleic acid sequences are possible which encode a Gene214-like protein or polypeptide. Some of these will have little homologyto the nucleotide sequences of any known or naturally-occurring Gene214-like gene but can be used to produce the proteins and polypeptidesof this invention by selection of combinations of nucleotide tripletsbased on codon choices. Such variants, while not hybridizable to anaturally-occurring Gene 214 gene, are contemplated within thisinvention.

The nucleic acids described herein are used in the methods of thepresent invention for production of proteins or polypeptides, throughincorporation into cells, tissues, or organisms. In one embodiment, DNAcontaining all or part of the coding sequence for a Gene 214polypeptide, or DNA which hybridizes to DNA having the sequence SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, and SEQ ID NO:10, isincorporated into a vector for expression of the encoded polypeptide insuitable host cells. The encoded polypeptide consisting of Gene 214, orits functional equivalent is capable of normal activity, such asprotecting the respiratory epithelium. The term “vector” as used hereinrefers to a nucleic acid molecule capable of replicating another nucleicacid to which it has been linked. A vector, for example, can be aplasmid.

Nucleic acids referred to herein as “isolated” are nucleic acidsseparated away from the nucleic acids of the genomic DNA or cellular RNAof their source of origin (e.g., as it exists in cells or in a mixtureof nucleic acids such as a library), and may have undergone furtherprocessing. “Isolated”, as used herein, refers to nucleic or amino acidsequences that are at least 60% free, preferably 75% free, and mostpreferably 90% free from other components with which they are naturallyassociated. “Isolated” nucleic acids (polynucleotides) include nucleicacids obtained by methods described herein, similar methods or othersuitable methods, including essentially pure nucleic acids, nucleicacids produced by chemical synthesis, by combinations of biological andchemical methods, and recombinant nucleic acids which are isolated.Nucleic acids referred to herein as “recombinant” are nucleic acidswhich have been produced by recombinant DNA methodology, including thosenucleic acids that are generated by procedures which rely upon a methodof artificial replication, such as the polymerase chain reaction (PCR)and/or cloning into a vector using restriction enzymes. “Recombinant”nucleic acids are also those that result from recombination events thatoccur through the natural mechanisms of cells, but are selected forafter the introduction to the cells of nucleic acids designed to allowor make probable a desired recombination event. Portions of the isolatednucleic acids which code for polypeptides having a certain function canbe identified and isolated by, for example, the method of Jasin, M., etal., U.S. Pat. No. 4,952,501.

A further embodiment of the invention is antisense nucleic acids oroligonucleotides which are complementary, in whole or in part, to atarget molecule comprising a sense strand, and can hybridize with thetarget molecule. The target can be DNA, or its RNA counterpart (i.e.,wherein T residues of the DNA are U residues in the RNA counterpart).When introduced into a cell, antisense nucleic acids or oligonucleotidescan inhibit the expression of the gene encoded by the sense strand orthe mRNA transcribed from the sense strand. Antisense nucleic acids canbe produced by standard techniques. See, for example, Shewmaker, et al.,U.S. Pat. No. 5,107,065.

In a particular embodiment, an antisense nucleic acid or oligonucleotideis wholly or partially complementary to and can hybridize with a targetnucleic acid (either DNA or RNA), wherein the target nucleic acid canhybridize to a nucleic acid having the sequence of the complement of thestrand in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, and SEQID NO:10. For example, an antisense nucleic acid or oligonucleotide canbe complementary to a target nucleic acid having the sequence shown asthe strand of the open reading frame of SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO: 8, and SEQ ID NO:10 or nucleic acid encoding afunctional equivalent of Gene 214, or to a portion of these nucleicacids sufficient to allow hybridization. A portion, for example asequence of 16 nucleotides, could be sufficient to inhibit expression ofthe protein. Or, an antisense nucleic acid or oligonucleotide,complementary to 5′ or 3′ untranslated regions, or overlapping thetranslation initiation codon (5′ untranslated and translated regions),of the Gene 214 gene, or a gene encoding a functional equivalent canalso be effective. In another embodiment, the antisense nucleic acid iswholly or partially complementary to and can hybridize with a targetnucleic acid which encodes a Gene 214 polypeptide.

In addition to the antisense nucleic acids of the invention,oligonucleotides can be constructed which will bind to duplex nucleicacid either in the gene or the DNA:RNA complex of transcription, to forma stable triple helix-containing or triplex nucleic acid to inhibittranscription and/or expression of a gene encoding Gene 214, or itsfunctional equivalent (Frank-Kamenetskii, M. D. and Mirkin, S. M. (1995)Ann. Rev. Biochem. 64:65-95.) Such oligonucleotides of the invention areconstructed using the base-pairing rules of triple helix formation andthe nucleotide sequence of the gene or mRNA for Gene 214. Theseoligonucleotides can block Gene 214-type activity in a number of ways,including prevention of transcription of the Gene 214 gene or by bindingto mRNA as it is transcribed by the gene.

The invention also relates to proteins or polypeptides encoded by thenovel nucleic acids described herein. The proteins and polypeptides ofthis invention can be isolated and/or recombinant. Proteins orpolypeptides referred to herein as “isolated” are proteins orpolypeptides purified to a state beyond that in which they exist incells. In a preferred embodiment, they are at least 10% pure; i.e., mostpreferably they are substantially purified to 80 or 90% purity.“Isolated” proteins or polypeptides include proteins or polypeptidesobtained by methods described infra, similar methods or other suitablemethods, and include essentially pure proteins or polypeptides, proteinsor polypeptides produced by chemical synthesis or by combinations ofbiological and chemical methods, and recombinant proteins orpolypeptides which are isolated. Proteins or polypeptides referred toherein as “recombinant” are proteins or polypeptides produced by theexpression of recombinant nucleic acids.

In a preferred embodiment, the protein or portion thereof has at leastone function characteristic of a Gene 214 protein or polypeptide, forexample, protective barrier to the respiratory epithelium activity inthe case of Gene 214 analogs, and/or antigenic function (e.g., bindingof antibodies that also bind to naturally occurring Gene 214polypeptide). As such, these proteins are referred to as analogs, andinclude, for example, naturally occurring Gene 214, variants (e.g.mutants) of those proteins and/or portions thereof. Such variantsinclude mutants differing by the addition, deletion or substitution ofone or more amino acid residues, or modified polypeptides in which oneor more residues are modified, and mutants comprising one or moremodified residues. The variant can have “conservative” changes, whereina substituted amino acid has similar structural or chemical properties,e.g., replacement of leucine with isoleucine. More infrequently, avariant can have “nonconservative” changes, e.g., replacement of aglycine with a tryptophan. Guidance in determining which amino acidresidues can be substituted, inserted, or deleted without abolishingbiological or immunological activity can be found using computerprograms well known in the art, for example, DNASTAR software (DNASTAR,Inc., Madison, Wis. 53715 U.S.A.).

A “portion” as used herein with regard to a protein or polypeptide,refers to fragments of that protein or polypeptide. The fragments canrange in size from 5 amino acid residues to all but one residue of theentire protein sequence. Thus, a portion or fragment can be at least 5,5-50, 50-100, 100-200, 200-400, 400-800, or more consecutive amino acidresidues of a Gene 214 protein or polypeptide, for example, SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 9 and SEQ ID NO: 11, or a variantthereof.

The invention also relates to isolated, synthesized and/or recombinantportions or fragments of a Gene 214 protein or polypeptide as describedabove. Polypeptide fragments of the enzyme can be made which have fullor partial function on their own, or which when mixed together (thoughfully, partially, or nonfunctional alone), spontaneously assemble withone or more other polypeptides to reconstitute a functional proteinhaving at least one functional characteristic of a Gene 214 proteinofthis invention.

The invention also concerns the use of the nucleotide sequence of thenucleic acids of this invention to identify DNA probes for Gene 214genes, PCR primers to amplify Gene 214 genes, nucleotide polymorphismsin Gene 214 genes, and regulatory elements of the Gene 214 genes.

Gene 214 was isolated by narrowly defining the region of chromosome12q23-qter 12q23-qter which was associated with airwayhyperresponsiveness and asthma. Gene 214 is also important in otherdiseases such as obesity and thus, there was a need to identify andisolate the gene.

To aid in the understanding of the specification and claims, thefollowing definitions are provided.

“Disorder region” refers to a portion of the human chromosome 12 boundedby the markers D12S2070 to the 12q telomere. A “disorder-associated”nucleic acid or “disorder-associated” polypeptide sequence refers to anucleic acid sequence that maps to region 12q23-qter and polypeptidesencoded therein. For nucleic acid sequences, this encompasses sequencesthat are homologous or complementary to the sequence, as well as“sequence-conservative variants” and “function-conservative variants.”For polypeptide sequences, this encompasses “function-conservativevariants.” Included are naturally-occurring mutations causative ofrespiratory diseases or obesity, such as but not limited to mutationswhich cause inappropriate expression (e.g., lack of expression,over-expression, expression in an inappropriate tissue type).“Sequence-conservative” variants are those in which a change of one ormore nucleotides in a given codon position results in no alteration inthe amino acid encoded at that position. “Function-conservative”variants are those in which a change in one or more nucleotides in agiven codon position results in a polypeptide sequence in which a givenamino acid residue in a polypeptide has been changed withoutsubstantially altering the overall conformation and function of thenative polypeptide, including, but not limited to, replacement of anamino acid with one having similar physico-chemical properties (such as,for example, acidic, basic, hydrophobic, and the like).“Function-conservative” variants also include analogs of a givenpolypeptide and any polypeptides that have the ability to elicitantibodies specific to a designated polypeptide.

“Nucleic acid or “polynucleotide” as used herein refers to purine- andpyrimidine-containing polymers of any length, either polyribonucleotidesor polydeoxyribonucleotide or mixed polyribo-polydeoxyribo nucleotides.This includes single- and double-stranded molecules, i.e., DNA-DNA,DNA-RNA and RNA-RNA hybrids, as well as “protein nucleic acids” (PNA)formed by conjugating bases to an amino acid backbone. This alsoincludes nucleic acids containing modified bases.

A “coding sequence” or a “protein-coding sequence” is a polynucleotidesequence capable of being transcribed into mRNA and/or capable of beingtranslated into a polypeptide. The boundaries of the coding sequence aretypically determined by a translation start codon at the 5′-terminus anda translation stop codon at the 3′-terminus.

A “complement” of a nucleic acid sequence as used herein refers to the“antisense” sequence that participates in Watson-Crick base-pairing withthe original sequence.

A “probe” refers to a nucleic acid or oligonucleotide that forms ahybrid structure with a sequence in a target region due tocomplementarily of at least one sequence in the probe with a sequence inthe target region.

Nucleic acids are “hybridizable” to each other when at least one strandof nucleic acid can anneal to another nucleic acid strand under definedstringency conditions. As is well known in the art, stringency ofhybridization is determined, e.g., by (a) the temperature at whichhybridization and/or washing is performed, and (b) the ionic strengthand polarity (e.g., formamide) of the hybridization and washingsolutions, as well as other parameters. Hybridization requires that thetwo nucleic acids contain substantially complementary sequences;depending on the stringency of hybridization, however, mismatches may betolerated. The appropriate stringency for hybridizing nucleic acidsdepends on the length of the nucleic acids and the degree ofcomplementarily, variables well known in the art.

An “immunogenic component”, is a moiety that is capable of eliciting ahumoral and/or cellular immune response in a host animal.

An “antigenic component” is a moiety that binds to its specific antibodywith sufficiently high affinity to form a detectable antigen-antibodycomplex.

A “sample” as used herein refers to a biological sample, such as, forexample, tissue or fluid isolated from an individual (including withoutlimitation plasma, serum, cerebrospinal fluid, lymph, tears, saliva,milk, pus, and tissue exudates and secretions) or from in vitro cellculture constituents, as well as samples obtained from e.g., alaboratory procedure.

“Gene” refers to a DNA sequence that encodes through its template ormessenger RNA a sequence of amino acids characteristic of a specificpeptide, polypeptide or protein. The term “gene” as used herein withreference to genomic DNA includes intervening, non-coding regions, aswell as regulatory regions, and can include 5′ and 3′ ends.

“Gene sequence” refers to a DNA molecule, including both a DNA moleculewhich contains a non-transcribed or non-translated sequence. The term isalso intended to include any combination of gene(s), gene fragment(s),non-transcribed sequence(s) or non-translated sequence(s) which arepresent on the same DNA molecule.

A gene sequence is “wild-type” if such sequence is usually found inindividuals unaffected by the disease or condition of interest. However,environmental factors and other genes can also play an important role inthe ultimate determination of the disease. In the context of complexdiseases involving multiple genes (“oligogenic disease”), the “wildtype” or normal sequence can also be associated with a measurable riskor susceptibility, receiving its reference status based on its frequencyin the general population.

A gene sequence is a “mutant” sequence if it differs from the wild-typesequence. In some cases, the individual carrying such gene has increasedsusceptibility toward the disease or condition of interest. In othercases, the “mutant” sequence might also refer to a sequence thatdecreases the susceptibility toward a disease or condition of interest,and thus acting in a protective manner. Also a gene is a “mutant” geneif too much (“overexpressed”) or too little (“underexpressed”) of suchgene is expressed in the tissues in which such gene is normallyexpressed, thereby causing the disease or condition of interest.

A gene sequence is a “variant” sequence if it is substantially similarin structure to either the entire gene or to a fragment of the gene.Both wild-type genes and mutant genes have variant sequences.

The sequences of the present invention may be derived from a variety ofsources including DNA, cDNA, synthetic DNA, synthetic RNA orcombinations thereof. Such sequences may comprise genomic DNA which mayor may not include naturally occurring introns. Moreover, such genomicDNA may be obtained in association with promoter regions or poly (A)sequences. The sequences, genomic DNA or cDNA may be obtained in any ofseveral ways. Genomic DNA can be extracted and purified from suitablecells by means well known in the art. Alternatively, mRNA can beisolated from a cell and used to produce cDNA by reverse transcriptionor other means.

“cDNA” refers to complementary or copy DNA produced from an RNA templateby the action of RNA-dependent DNA polymerase (reverse transcriptase).Thus, a “cDNA clone” means a duplex DNA sequence complementary to an RNAmolecule of interest, carried in a cloning vector or PCR amplified. Thisterm includes genes from which the intervening sequences have beenremoved.

“Recombinant DNA” means a molecule that has been recombined by in vitrosplicing/and includes cDNA or a genomic DNA sequence.

“Cloning” refers to the use of in vitro recombination techniques toinsert a particular gene or other DNA sequence into a vector molecule.In order to successfully clone a desired gene, it is necessary to usemethods for generating DNA fragments, for joining the fragments tovector molecules, for introducing the composite DNA molecule into a hostcell in which it can replicate, and for selecting the clone having thetarget gene from amongst the recipient host cells.

“cDNA library” refers to a collection of recombinant DNA moleculescontaining cDNA inserts which together comprise the entire genome of anorganism. Such a cDNA library can be prepared by methods known to oneskilled in the art and described by, for example, Cowell and Austin,“cDNA Library Protocols,” Methods in Molecular Biology (1997).Generally, RNA is first isolated from the cells of an organism fromwhose genome it is desired to clone a particular gene.

“Cloning vehicle” refers to a plasmid or phage DNA or other DNA sequencewhich is able to replicate in a host cell. The cloning vehicle ischaracterized by one or more endonuclease recognition sites at whichsuch DNA sequences may be cut in a determinable fashion without loss ofan essential biological function of the DNA, which may contain a markersuitable for use in the identification of transformed cells.

“Expression control sequence” refers to a sequence of nucleotides thatcontrol or regulate expression of structural genes when operably linkedto those genes. These include, for example, the lac systems, the trpsystem, major operator and promoter regions of the phage lambda, thecontrol region of fd coat protein and other sequences known to controlthe expression of genes in prokaryotic or eukaryotic cells. Expressioncontrol sequences will vary depending on whether the vector is designedto express the operably linked gene in a prokaryotic or eukaryotic host,and may contain transcriptional elements such as enhancer elements,termination sequences, tissue-specificity elements and/or translationalinitiation and termination sites.

“Expression vehicle” refers to a vehicle or vector similar to a cloningvehicle but which is capable of expressing a gene which has been clonedinto it, after transformation into a host. The cloned gene is usuallyplaced under the control of (i.e., operably linked to) an expressioncontrol sequence.

“Operably linked” means that the promoter controls the initiation ofexpression of the gene. A promoter is operably linked to a sequence ofproximal DNA if upon introduction into a host cell the promoterdetermines the transcription of the proximal DNA sequence(s) into one ormore species of RNA. A promoter is operably linked to a DNA sequence ifthe promoter is capable of initiating transcription of that DNAsequence.

“Host” includes prokaryotes and eukaryotes. The term includes anorganism or cell that is the recipient of a replicable expressionvehicle.

“Amplification of nucleic acids” refers to methods such as polymerasechain reaction (PCR), ligation amplification (or ligase chain reaction,LCR) and amplification methods based on the use of Q-beta replicase.These methods are well known in the art and described, for example, inU.S. Pat. Nos. 4,683,195 and 4,683,202. Reagents and hardware forconducting PCR are commercially available. Primers useful for amplifyingsequences from the disorder region are preferably complementary to, andpreferably hybridize specifically to, sequences in the 12q23-qter regionor in regions that flank a target region therein. Gene 214 generated byamplification may be sequenced directly. Alternatively, the amplifiedsequence(s) may be cloned prior to sequence analysis.

“Antibodies” refer to polyclonal and/or monoclonal antibodies andfragments thereof, and immunologic binding equivalents thereof, that canbind to asthma proteins and fragments thereof or to nucleic acidsequences from the 12q23-qter region, particularly from the asthma locusor a portion thereof. The term antibody is used both to refer to ahomogeneous molecular entity, or a mixture such as a serum product madeup of a plurality of different molecular entities. Proteins may beprepared synthetically in a protein synthesizer and coupled to a carriermolecule and injected over several months into rabbits. Rabbit sera istested for immunoreactivity to the protein or fragment. Monoclonalantibodies may be made by injecting mice with the proteins, or fragmentsthereof. Monoclonal antibodies will be screened by ELISA and tested forspecific immunoreactivity with protein or fragments thereof. (Harlow etal, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1988.) These antibodies will be useful assays aswell as pharmaceuticals.

A nucleic acid or fragment thereof is “substantially homologous” or“substantially similar” to another if, when optimally aligned (withappropriate nucleotide insertions and/or deletions) with the othernucleic acid (or its complementary strand), there is nucleotide sequenceidentity in at least about 60% of the nucleotide bases, usually at leastabout 70%, more usually at least about 80%, preferably at least about90%, and more preferably at least about 95-98% of the nucleotide bases.

Alternatively, substantial homology or similarity exists when a nucleicacid or fragment thereof will hybridize, under selective hybridizationconditions, to another nucleic acid (or a complementary strand thereof).Selectivity of hybridization exists when hybridization which issubstantially more selective than total lack of specificity occurs.Typically, selective hybridization will occur when there is at leastabout 55% homology over a stretch of at least about nine or morenucleotides, preferably at least about 65%, more preferably at leastabout 75%, and most preferably at least about 90%. (See, M. Kanehisa,1984, Nucleic Acids Res., 12(1 Pt 1):203-13; M. Kanehisa et al., 1984,Nucleic Acids Res., 12(1 Pt 1):149-58; M. Kanehisa et al., 1984, NucleicAcids Res. 12(1 Pt 1):417-28). The length of homology comparison, asdescribed, may be over longer stretches, and in certain embodiments willoften be over a stretch of at least about 14 nucleotides, usually atleast about 20 nucleotides, more usually at least about 24 nucleotides,typically at least about 28 nucleotides, more typically at least about32 nucleotides, and preferably at least about 36 or more nucleotides.

Technical and scientific terms used herein have the meanings commonlyunderstood by one of ordinary skill in the art to which the presentinvention pertains, unless otherwise defined. Reference is made hereinto various methodologies known to those of skill in the art.Publications and other materials setting forth such known methodologiesto which reference is made are incorporated herein by reference in theirentireties as though set forth in full. Standard reference works settingforth the general principles of recombinant DNA technology includeSambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory Press, Planview, N.Y. (1989); Kaufman, P.B., et al., Eds., Handbook of Molecular and Cellular Methods in Biologyand Medicine, CRC Press, Boca Raton (1995); McPherson, M. J., Ed.,Directed Mutagenesis: A Practical Approach, IRL Press, Oxford (1991);Jones, J., Amino Acid and Peptide Synthesis, Oxford SciencePublications, Oxford (1992); Austen, B. M. and Westwood, O. M. R.,Protein Targeting and Secretion, IRL Press, Oxford (1991); DNA Cloning,Volumes I and II (D. N Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed, 1984); Nucleic Acid Hybridization (B. D. Hames & S. J.Higgins eds. 1984); the series, Methods in Enzymology (Academic Press,Inc.), particularly Vol. 154 and Vol. 155 (Wu and Grossman, eds.); PCR-APractical Approach (McPherson, Quirke, and Taylor, eds., 1991);Transcription and Translation, 1984 (Hames and Higgins eds.); AnimalCell Culture, 1986 (R. I. Freshney ed.); Immobilized Cells and Enzymes,1986 (IRL Press); Perbal, 1984, A Practical Guide to Molecular Cloning;Gene Transfer Vectors for Mammalian Cells, 1987 (J. H. Miller and M. P.Calos eds., Cold Spring Harbor Laboratory); Martin J. Bishop, ed., Guideto Human Genome Computing, 2d Edition, Academic Press, San Diego, Calif.(1998); and Leonard F. Peruski, Jr., and Anne Harwood Peruski, TheInternet and the New Biology: Tools for Genomic and Molecular Research,American Society for Microbiology, Washington, D.C. (1997). Standardreference works setting forth the general principles of immunologyinclude Sell, S., Immunology, Immunopathology & Immunity, 5th Ed.,Appleton & Lange, Publ., Stamford, Conn. (1996); Male, D., et al.,Advanced Immunology, 3d Ed., Times Mirror Int'l Publishers Ltd., Publ.,London (1996); Stites, D. P., and Ten, A. I., Basic and ClinicalImmunology, 7th Ed., Appleton & Lange, Publ., Norwalk, Conn. (1991); andAbbas, A. K., et al., Cellular and Molecular Immunology, W. B. SaundersCo., Publ., Philadelphia, Pa. (1991). Any suitable materials and/ormethods known to those of skill can be utilized in carrying out thepresent invention; however, preferred materials and/or methods aredescribed. Materials, reagents and the like to which reference is madein the following description and examples are obtainable from commercialsources, unless otherwise noted.

The nucleic acids of the invention may be isolated directly from cells.Alternatively, the polymerase chain reaction (PCR) method can be used toproduce the nucleic acids of the invention, using either chemicallysynthesized strands or genomic material as templates. Primers used forPCR can be synthesized using the sequence information provided hereinand can further be designed to introduce appropriate new restrictionsites, if desirable, to facilitate incorporation into a given vector forrecombinant expression.

The invention also provides vectors comprising the disorder-associatedsequences or derivatives or fragments thereof and host cells for theproduction of purified proteins. A large number of vectors, includingplasmid and fungal vectors, have been described for replication and/orexpression in a variety of eukaryotic and prokaryotic hosts, and may beused for gene therapy as well as for simple cloning or proteinexpression.

Using the information provided in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO: 8, and SEQ ID NO:10, one skilled in the art will be able toclone and sequence all representative nucleic acids of interest,including nucleic acids encoding complete protein-coding sequences. Itis to be understood that non-protein-coding sequences contained withinSEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, and SEQ ID NO:10and the genomic sequence of SEQ ID NO:1 are also within the scope of theinvention. Such sequences include, without limitation, sequencesimportant for replication, recombination, transcription and translation.Non-limiting examples include promoters and regulatory binding sitesinvolved in regulation of gene expression, and 5′- and 3′-untranslatedsequences (e.g., ribosome-binding sites) that form part of mRNAmolecules.

The nucleic acids of the present invention find use as primers andtemplates for the recombinant production of disorder-associated peptidesor polypeptides, for chromosome and gene mapping, to provide antisensesequences, for tissue distribution studies, to locate and obtain fulllength genes, to identify and obtain homologous sequences (wild-type andmutants), and in diagnostic applications.

Polypeptides according to the invention are at least five or moreresidues in length. Preferably, the polypeptides comprise at least about12, more preferably at least about 20 and most preferably at least about30 such residues. Nucleic acids comprising protein-coding sequences canbe used to direct the expression of asthma-associated polypeptides inintact cells or in cell-free translation systems. The known geneticcode, tailored if desired for more efficient expression in a given hostorganism, can be used to synthesize oligonucleotides encoding thedesired amino acid sequences. The resulting oligonucleotides can beinserted into an appropriate vector and expressed in a compatible hostorganism.

The polypeptides of the present invention, includingfunction-conservative variants, may be isolated from wild-type or mutantcells, or from heterologous organisms or cells (e.g., bacteria, fungi,yeast, insect, plant, and mammalian cells) in which adisorder-associated protein-coding sequence has been introduced andexpressed. Furthermore, the polypeptides may be part of recombinantfusion proteins. The polypeptides can also, advantageously, be madeusing cell-free protein synthesis systems or by synthetic chemistry.Polypeptides may be chemically synthesized by commercially availableautomated procedures, including, without limitation, exclusive solidphase synthesis, partial solid phase methods, fragment condensation orclassical solution synthesis.

Methods for polypeptide purification are well-known in the art,including, without limitation, preparative disc-gel electrophoresis,isoelectric focusing, HPLC, reversed-phase HPLC, gel filtration, ionexchange and partition chromatography, and countercurrent distribution.For some purposes, it is preferable to produce the polypeptide in arecombinant system in which the disorder-associated protein contains anadditional sequence tag that facilitates purification. Alternatively,antibodies produced against an disorder-associated protein or againstpeptides derived therefrom can be used as purification reagents. Otherpurification methods are possible.

The present invention also encompasses derivatives and homologies ofdisorder-associated polypeptides. For some purposes, nucleic acidsequences encoding the peptides may be altered by substitutions,additions, or deletions that provide for functionally equivalentmolecules, i.e., function-conservative variants.

The isolated polypeptides may be modified by, for example,phosphorylation, sulfation, acylation, or other protein modifications.They may also be modified with a label capable of providing a detectablesignal, either directly or indirectly, including, but not limited to,radioisotopes and fluorescent compounds.

Both the naturally occurring and recombinant forms of the polypeptidesof the invention can advantageously be used to screen compounds forbinding activity. Many methods of screening for binding activity areknown by those skilled in the art and may be used to practice theinvention. Several methods of automated assays have been developed inrecent years so as to permit screening of tens of thousands of compoundsin a short period of time. Such high-throughput screening methods areparticularly preferred. The use of high-throughput screening assays totest for inhibitors is greatly facilitated by the availability of largeamounts of purified polypeptides, as provided by the invention. Thepolypeptides of the invention also find use as therapeutic agents aswell as antigenic components to prepare antibodies.

The polypeptides of this invention find use as immunogenic componentsuseful as antigens for preparing antibodies by standard methods. It iswell known in the art that immunogenic epitopes generally contain atleast about five amino acid residues, Ohno et al., 1985, Proc. Natl.Acad. Sci. USA 82:2945. Therefore, the immunogenic components of thisinvention will typically comprise at least five amino acid residues ofthe sequence of the complete polypeptide chains. Preferably, they willcontain at least 7, and most preferably at least about 10 amino acidresidues or more to ensure that they will be immunogenic. Whether agiven component is immunogenic can readily be determined by routineexperimentation Such immunogenic components can be produced byproteolytic cleavage of larger polypeptides or by chemical synthesis orrecombinant technology and are thus not limited by proteolytic cleavagesites. The present invention thus encompasses antibodies thatspecifically recognize asthma-associated immunogenic components.

Antibodies according to the present invention include polyclonal andmonoclonal antibodies. The antibodies may be elicited in an animal hostby immunization with disorder-associated immunogenic components or maybe formed by in vitro immunization (sensitization) of immune cells. Theimmunogenic components used to elicit the production of antibodies maybe isolated from cells or chemically synthesized. The antibodies mayalso be produced in recombinant systems programmed with appropriateantibody-encoding DNA. Alternatively, the antibodies may be constructedby biochemical reconstitution of purified heavy and light chains. Theantibodies include hybrid antibodies, chimeric antibodies, and univalentantibodies. Also included are Fab fragments, including Fab¹ and Fab(ab)²fragments of antibodies.

These antibodies, whether polyclonal or monoclonal, can be used, e.g.,in an immobilized form bound to a solid support by well known methods,to purify the immunogenic components and disorder-associatedpolypeptides by immunoaffinity chromatography. Antibodies against theimmunogenic components can also be used, unlabeled or labeled bystandard methods, as the basis for immunoassays, i.e., as diagnosticreagents.

Hybridomas of the invention used to make monoclonal antibodies againstthe immunogenic components of the invention are produced by well-knowntechniques. Usually, the process involves the fusion of an immortalizingcell line with a B-lymphocyte that produces the desired antibody.Alternatively, non-fusion techniques for generating immortalantibody-producing cell lines are possible, and come within the purviewof the present invention, e.g., virally-induced transformation, Casaliet al., 1986, Science 234:476. Immortalizing cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine, and human origin. Most frequently, rat or mouse myeloma celllines are employed as a matter of convenience and availability.

Hybridomas are selected by standard procedures, such as HAT(hypoxanthine-aminopterin-thymidine) selection. From among thesehybridomas, those secreting the desired antibody are selected byassaying their culture medium by standard immunoassays, such as Westernblotting, ELISA (enzyme-linked immunosorbent assay), RtA(radioimmunoassay), or the like. Antibodies are recovered from themedium using standard protein purification techniques, Tijssen, 1985,Practice and Theory of Enzyme Immunoassays, Elsevier, Amsterdam.

I. Localization of an Asthma Locus on Chromosome 12q23-qter and theCharacterization of a Candidate Gene Within the Region

To identify genes in the region on 12q23-qter, a set of bacterialartificial chromosome (BAC) clones containing this chromosomal regionwas identified. The BAC clones served as a template for genomic DNAsequencing and serve as reagents for identifying coding sequences bydirect cDNA selection. Genomic sequencing and direct cDNA selection wereused to characterize DNA from 12q23-qter.

When a gene has been genetically localized to a specific chromosomalregion, the genes in this region can be characterized at the molecularlevel by a series of steps that include: cloning of the entire region ofDNA in a set of overlapping clones (physical mapping), characterizationof genes encoded by these clones by a combination of direct cDNAselection, exon trapping and DNA sequencing (gene identification), andidentification of mutations in these genes by comparative DNA sequencingof affected and unaffected members of the kindred and/or in unrelatedaffected individuals and unrelated unaffected controls (mutationanalysis).

Physical mapping is accomplished by screening libraries of human DNAcloned in vectors that are propagated in a host such as E. coli, usinghybridization or PCR assays from unique molecular landmarks in thechromosomal region of interest. To generate a physical map of thedisorder region, a library of human DNA cloned in BACs was screened witha set of overgo markers that had been previously mapped to chromosome12q23-qter by the efforts of the Human Genome Project. Overgos areunique molecular landmarks in the human genome that can be assayed byhybridization. Through the combined efforts of the Human Genome Project,the location of thousands of overgos on the twenty-two autosomes and twosex chromosomes has been determined. For a positional cloning effort,the physical map is tied to the genetic map because the markers used forgenetic mapping can also be used as overgos for physical mapping. Byscreening a BAC library with a combination of overgos derived fromgenetic markers, genes, and random DNA fragments, a physical mapcomprised of overlapping clones representing all of the DNA in achromosomal region of interest can be assembled.

BACs are cloning vectors for large (80 kilobase to 200 kilobase)segments of human or other DNA that are propagated in E. coli. Toconstruct a physical map using BACs, a library of BAC clones is screenedso that individual clones harboring the DNA sequence corresponding to agiven overgo or set of overgos are identified. Throughout most of thehuman genome, the overgo markers are spaced approximately 20 to 50kilobases apart, so that an individual BAC clone typically contains atleast two overgo markers. In addition, the BAC libraries that werescreened contain enough cloned DNA to cover the human genome twelvetimes over. Therefore, an individual overgo typically identifies morethan one BAC clone. By screening a twelve-fold coverage BAC library witha series of overgo markers spaced approximately 50 kilobases apart, aphysical map consisting of a series of overlapping contiguous BACclones, i.e., BAC “contigs,” can be assembled for any region of thehuman genome. This map is closely tied to the genetic map because manyof the overgo markers used to prepare the physical map are also geneticmarkers.

When constructing a physical map, it often happens that there are gapsin the overgo map of the genome that result in the inability to identifyBAC clones that are overlapping in a given location. Typically, thephysical map is first constructed from a set of overgos identifiedthrough the publicly available literature and World Wide Web resources.The initial map consists of several separate BAC contigs that areseparated by gaps of unknown molecular distance. To identify BAC clonesthat fill these gaps, it is necessary to develop new overgo markers fromthe ends of the clones on either side of the gap. This is done bysequencing the terminal 200 to 300 base pairs of the BACs flanking thegap, and developing a PCR or hybridization based assay. If the terminalsequences are demonstrated to be unique within the human genome, thenthe new overgo can be used to screen the BAC library to identifyadditional BACs that contain the DNA from the gap in the physical map.To assemble a BAC contig that covers a region the size of the disorderregion (6,000,000 or more base pairs), it is necessary to develop newovergo markers from the ends of a number of clones.

After building a BAC contig, this set of overlapping clones serves as atemplate for identifying the genes encoded in the chromosomal region.Gene identification can be accomplished by many methods. Three methodsare commonly used: (1) a set of BACs selected from the BAC contig torepresent the entire chromosomal region can be sequenced, andcomputational methods can be used to identify all of the genes, (2) theBACs from the BAC contig can be used as a reagent to clone cDNAscorresponding to the genes encoded in the region by a method termeddirect cDNA selection, or (3) the BACs from the BAC contig can be usedto identify coding sequences by selecting for specific DNA sequencemotifs in a procedure called exon trapping. The present inventionincludes Gene 214 identified by the first two methods.

To sequence the entire BAC contig representing the disorder region, aset of BACs can be chosen for subcloning into plasmid vectors andsubsequent DNA sequencing of these subclones. Since the DNA cloned inthe BACs represents genomic DNA, this sequencing is referred to asgenomic sequencing to distinguish it from cDNA sequencing. To initiatethe genomic sequencing for a chromosomal region of interest, severalnon-overlapping BAC clones are chosen. DNA for each BAC clone isprepared, and the clones are sheared into random small fragments whichare subsequently cloned into standard plasmid vectors such as pUC18. Theplasmid clones are then grown to propagate the smaller fragments, andthese are the templates for sequencing. To ensure adequate coverage andsequence quality for the BAC DNA sequence, sufficient plasmid clones aresequenced to yield three-fold coverage of the BAC clone. For example, ifthe BAC is 100 kilobases long, then phagemids are sequenced to yield 300kilobases of sequence. Since the BAC DNA was randomly sheared prior tocloning in the phagemid vector, the 300 kilobases of raw DNA sequencecan be assembled by computational methods into overlapping DNA sequencestermed sequence contigs. For the purposes of initial gene identificationby computational methods, three-fold coverage of each BAC is sufficientto yield twenty to forty sequence contigs of 1000 base pairs to 20,000base pairs.

The sequencing strategy employed in this invention was to initiallysequence “seed” BACs from the BAC contig in the disorder region. Thesequence of the “seed” BACs was then used to identify minimallyoverlapping BACs from the contig, and these were subsequently sequenced.In this manner, the entire candidate region can be sequenced, withseveral small sequence gaps left in each BAC. This sequence serves asthe template for computational gene identification. One method forcomputational gene identification is to compare the sequence of BACcontig to publicly available databases of cDNA and genomic sequences,e.g. unigene, dbEST, genbank. These comparisons are typically done usingthe BLAST family of computer algorithms and programs (Altschul et al, J.Mol. Biol., 215:403-410 (1990)). The BAC sequence can also be translatedinto protein sequence, and the protein sequence can be used to searchpublicly available protein databases, using a version of BLAST designedto analyze protein sequences (Altshul et al, Nucl. Acids Res.,25:3389-3402 (1997)). Another method is to use computer algorithms suchas MZEF (Zhang, Proc. Natl. Acad. Sci., 94:565-568 (1997)), GRAIL(Uberbacher et al, Methods Enzymol., 266:259-281 (1996)), and Genscan(Burge and Karlin, J. Mol. Biol., 268:78-94) which predicts the locationof exons in the sequence based on the presence of specific DNA sequencemotifs that are common to all exons, as well as the presence of codonusage typical of human protein encoding sequences.

In addition to identifying genes by computational methods, genes werealso identified by direct cDNA selection (Del Mastro and Lovett, Methodsin Molecular Biology, Humana Press Inc., NJ (1996)). In direct cDNAselection, cDNA pools from tissues of interest are prepared, and BACsfrom the candidate region are used in a liquid hybridization assay tocapture the cDNAs which base pair to coding regions in the BAC. In themethods described herein, the cDNA pools were created from severaldifferent tissues by random priming and oligo dT priming the firststrand cDNA from polyA RNA, synthesizing the second strand cDNA bystandard methods, and adding linkers to the ends of the cDNA fragments.The linkers are used to amplify the cDNA pools. BAC clones from thedisorder region identified by screening the RPCI-11 BAC library (P.deJong, Russell Park Cancer Institute) were used as a template forinitiating DNA synthesis to create a biotin labeled copy of BAC DNA. Thebiotin labelled copy of the BAC DNA is then denatured and incubated withan excess of the PCR amplified, linkered cDNA pools which have also beendenatured. The BAC DNA and cDNA are allowed to anneal in solution, andheteroduplexes between the BAC and the cDNA are isolated usingstreptavidin coated magnetic beads. The cDNAs that are captured by theBAC are then amplified using primers complimentary to the linkersequences, and the hybridization/selection process is repeated for asecond round. After two rounds of direct cDNA selection, the cDNAfragments are cloned, and a library of these direct selected fragmentsis created.

The cDNA clones isolated by direct selection are analyzed by twomethods. Since a pool of BACs from the disorder region is used toprovide the genomic target DNA sequence, the cDNAs must be mapped to BACgenomic clones to verify their chromosomal location. This isaccomplished by arraying the cDNAs in microtiter dishes, and replicatingtheir DNA in high density grids. Individual genomic clones known to mapto the region are then hybridized to the grid to identify directselected cDNAs mapping to that region. cDNA clones that are confirmed tocorrespond to individual BACs are sequenced. To determine whether thecDNA clones isolated by direct selection share sequence identity orsimilarity to previously identified genes, the DNA and protein codingsequences are compared to publicly available databases using the BLASTfamily of programs.

The combination of genomic DNA sequence and cDNA sequence provided byBAC sequencing and by direct cDNA selection yields an initial list ofputative genes in the region. The genes in the region were allcandidates for the asthma locus. To further characterize each gene,Northern blots were performed to determine the size of the transcriptcorresponding to each gene, and to determine which putative exons weretranscribed together to make an individual gene. For Northern blotanalysis of each gene, probes were prepared from direct selected cDNAclones or by PCR amplifying specific fragments from genomic DNA, cDNA orfrom the BAC encoding the putative gene of interest. The Northern blotsgave information on the size of the transcript and the tissues in whichit was expressed. For transcripts which were not highly expressed, itwas sometimes necessary to perform a reverse transcription PCR assayusing RNA from the tissues of interest as a template for the reaction.

Gene identification by computational methods and by direct cDNAselection provides unique information about the genes in a region of achromosome. When genes are identified, then it is possible to examinedifferent individuals for mutations in each gene. Variants in genesequences between individuals can be inherited allelic differences orcan arise from mutations in the individuals. Gene sequence variants areclinically important in that they can affect drug action on such gene.Most drugs elicit a safe response in only a fraction of individuals, anddrugs are commonly administered to patients with no certainty that theywill be safe and effective. Many important drugs are effective in only30-40% of patients for whom the drug is prescribed, and virtually alldrugs cause adverse events in some individuals. Identification ofmutations in disorder genes in different individuals will enable acorrelation between the safety and efficacy of drug therapies used totreat lung diseases and the genotypes of the treated individuals. Thiscorrelation enables health care providers to prescribe a drug regimenwhich is most appropriate for the individual patient rather than tryingdifferent drug regimens in turn until a successful drug is identified.Identification of variants in disorder genes will also have a benefitduring the development of new drugs for the treatment of lung diseases,as the ability to correlate genetic variation with the efficacy of newcandidate drugs will enhance lead optimization and increase theefficiency and success rate of new drug approvals.

A. Family Collection

A critical component of any disease gene search is the careful selectionand phenotyping of family resources. The family collection utilized inthis study consists of 421 Caucasian affected sibling (“sib”) pairsfamilies collected in the United States and the United Kingdom, as wellas an additional 63 Caucasian families from the United Kingdom collectedunder different ascertainment criteria.

The affected sibling (or “sib”) pair families in the United Statescollection were Caucasian families with two affected siblings that wereidentified through both private practice and community physicians.Advertising was also used to identify candidates. A total of 98 familieswere collected in Kansas, Nebraska, and Southern California. In theUnited Kingdom collection, 323 families were identified throughphysicians' registers in a region surrounding Southampton and includingthe Isle of Wight.

Families were included in the study if they met all of the followingcriteria: (1) the biological mother and biological father were Caucasianand agreed to participate in the study, (2) at least two biologicalsiblings were alive, each with a current physician diagnosis of asthma,and 5 to 21 years of age, and (3) the two siblings were currently takingasthma medications on a regular basis. This included regular,intermittent use of inhaled or oral bronchodilators and regular use ofcromolyn, theophylline, or steroids.

Families were excluded from the study if they met any one of thefollowing criteria: (1) both parents were affected (i.e., with a currentdiagnosis of asthma, having asthma symptoms, or on asthma medications atthe time of the study), or (2) any of the siblings to be included in thestudy was less than 5 years of age, or (3) any asthmatic family memberto be included in the study was taking beta-blockers at the time of thestudy or (4) any family member had congenital or acquired pulmonarydisease at birth (e.g. cystic fibrosis) history of serious cardiacdisease (myocardial infarction) or any history of serious pulmonarydisease (e.g. emphysema) or (5) pregnant.

An additional 63 families from the United Kingdom were utilized from anearlier collection effort with different ascertainment criteria. Thesefamilies were recruited either: 1) without reference to asthma and atopyor 2) by having at least one family member or at least two familymembers affected with asthma. The randomly ascertained samples wereidentified from general practitioner registers in the Southampton area.For the families with affected members, the probands were recruited fromhospital based clinics in Southampton. The phenotypic and genotypic datainformation for 17 markers for 21 of these 63 families was obtained fromthe website:http://cedar.genetics.soton.ac.uk/pub/PROGRAMS/BETA/data/bet12.ped.

B. Genome Scan

In order to identify chromosomal regions linked to asthma, theinheritance pattern of alleles from genetic markers spanning the genomewas assessed on the collected family resources. As described above,combining these results with the segregation of the asthma phenotype inthese families allows the identification of genetic markers that aretightly linked to asthma, thus providing an indication of the locationof genes predisposing affected individuals to asthma. The followingdiscussion describes the protocol used to assess the genotypes of thecollected population using genetic markers spanning the entire genome.

Genotypes of PCR amplified simple sequence microsatellite geneticlinkage markers were determined using ABI model 377 AutomatedSequencers. Microsatellite markers comprising a variation of a humanlinkage mapping panel as released from the Cooperative Human LinkageCenter (CHLC), also known as the Weber lab screening set version 8, wereobtained from Research Genetics Inc. (Huntsville, Al.) in thefluorescent dye-conjugated form (Dubovsky et al., Hum. Mol. Genet.March; 4(3):449-452 (1995)).

Our variation of the Weber 8 screening set consists of 529 markers withan average spacing of 6.87 cM (autosomes only) and 6.98 cM (allchromosomes). Eighty-nine percent of the markers consist of either tri-or tetra-nucleotide microsatellites. In addition, there exist no gaps inchromosomal coverage greater than 17.5 cM.

Study subject genomic DNA (5 μl; 4.5 ng/μl) was amplified in a 10 μl PCRreaction using AmpliTaq Gold DNA polymerase (0.225 U) and containing thefinal reaction components: 1×PCR buffer (80 mM (NH₄)₂SO₄, 30 mM Tris-HCl(pH 8.8), 0.5% Tween-20), 200 μM each dATP, dCTP, dGTP and dTTP, 1.5-3.5μM MgCl₂ and 250 μM forward and reverse PCR primers. PCR reactions wereset up in 192 well plates (Costar) using a Tecan Genesis 150 roboticworkstation equipped with a refrigerated deck. PCR reactions wereoverlaid with 20 μl mineral oil, and thermocycled on an MJ ResearchTetrad DNA Engine equipped with four 192 well heads under the followingconditions: 92° C. for 3 min, 6 cycles of 92° C. 30 sec, 56° C. 1 min,72° C. 45 sec, followed by 20 cycles of 92° C. 30 sec, 55° C. 1 min, 72°C. 45 sec and a 6 min incubation at 72° C. PCR products of 8-12microsatellite markers were subsequently pooled using a Tecan Genesis200 robotic workstation into two 96 well microtitre plates (2.0 μl PCRproduct from TET and FAM labeled markers, 3.01 HEX labeled markers) andbrought to a final volume of 25 μl with H₂0. 1.9 μl of pooled PCRproduct was transferred to a loading plate and combined with 3.0 μlloading buffer (loading buffer is 2.51 formamide/blue dextran (9.0mg/ml), 0.5 μl GS-500 TAMRA labeled size standard, Perkin-Elmer/ABIdivision). Samples were denatured in the loading plate for 4 min at 95°C., placed on ice for 2 min, and electrophoresed in a 5% denaturingpolyacrylamide gel (FMC on the ABI 377XL). Samples (0.8 μl) were loadedusing an 8 channel Hamilton Syringe pipettor.

Each gel consisted of 62 study subjects and 2 control subjects (CEPHparents ID #1331-01 and 1331-02, Coriell Cell Repository, Camden, N.J.).Genotyping gels were scored in duplicate by investigators blind topatient identity and affection status using GENOTYPER analysis softwareV 1.1.12 (ABI Division, Perkin Elmer Corporation). Nuclear families wereloaded onto the gel with the parents flanking the siblings to facilitateerror detection. Data with allele peak amplitude less than 100, asdetected by GENESCAN analysis software V 2.0.2 (ABI Division, PerkinElmer Corporation), were either left unscored or rerun.

The final tables obtained from the Genotyper output for each gelanalysed were imported into a Sybase Database. Allele calling (binning)was performed using the SYBASE version of the ABAS software (Ghosh etal, Genome Research 7:165-178 (1997)). Offsize bins were checkedmanually and incorrect calls were corrected or blanked. The binnedalleles were then imported into the program MENDEL (Lange et al.,Genetic Epidemiology, 5, 471 (1988)) for inheritance checking using theUSERM13 subroutine (Boehnke et al, AM. J. Hum. Genet. 48:22-25 (1991)).Non-inheritance was investigated by examining the genotyping traces andonce all discrepancies were resolved, the subroutine USERM13 was used toestimate allele frequencies.

C. Linkage Analysis

Linkage analysis is possible because of the nature of inheritance ofchromosomes from parents to offspring. During meiosis, the two parentalhomologues pair to guide their proper separation to daughter cells.While they are lined up and paired, the two homologues exchange piecesof the chromosomes, in an event called “crossing over” or“recombination.” The resulting chromosomes contain parts that originatefrom both parental homologues. The closer together two sequences are onthe chromosome, the less likely that a recombination event will occurbetween them, and the more closely linked they are. Data obtained fromthe different families are combined and analyzed together by a computerusing statistical methods. The result is information indicating theevidence for linkage between the genetic markers used and a diseasesusceptibility locus. A recombination frequency of 1% is equivalent toapproximately 1 map unit, a relationship that holds up to frequencies ofabout 20% or 20 cM. Furthermore, 1 centiMorgan (cM) is roughlyequivalent to 1,000 kb of DNA.

The entire human genome is 3,300 cM long. In order to find an unknowndisease gene within 5-10 cM of a marker locus, the whole human genomecan be searched with roughly 330 informative marker loci spaced atapproximately 10 cM intervals (Botstein et al, Am. J. Hum. Genet.,32:314-331 (1980)). The reliability of linkage results is established byusing a number of statistical methods. The methods most commonly usedfor the detection by linkage analysis of oligogenes involved in theetiology of a complex trait are non-parametric or model-free methodswhich have been implemented into the computer programs MAPMAKER/SIBS(Kruglyak L & Lander E S, Am J Hum Genet. 57:439-454, 1995) andGENEHUNTER (Kruglyak L et al., Am J Hum Genet. 58:1347-1363, 1996).Linkage analysis is performed by typing members of families withmultiple affected individuals at a given marker locus and evaluating ifthe affected members (excluding parent-offspring pairs) share alleles atthe marker locus that are identical by descent (IBD) more often thanexpected by chance alone. As a result of the rapid advances in mappingthe human genome over the last few years, and concomitant improvementsin computer methodology, it has become feasible to carry out linkageanalyses using multi-point data. Multi-point analysis provides asimultaneous analysis of linkage between the trait and several linkedgenetic markers, when the recombination distance among the markers isknown. A LOD score statistic is computed at multiple locations along achromosome to measure the evidence that a susceptibility locus islocated nearby. A LOD score is the logarithm base 10 of the ratio of thelikelihood that a susceptibility locus exists at a given location to thelikelihood that no susceptibility locus is located there. By convention,when testing a single marker, a total LOD score greater than +3.0 (thatis, odds of linkage being 1,000 times greater than odds of no linkage)is considered to be significant evidence for linkage.

Multi-point analysis is advantageous for two reasons. First, theinformativeness of the pedigrees is usually increased. Each pedigree hasa certain amount of potential information, dependent on the number ofparents heterozygous for the marker loci and the number of affectedindividuals in the family. However, few markers are sufficientlypolymorphic as to be informative in all those individuals. If multiplemarkers are considered simultaneously, then the probability of anindividual being heterozygous for at least one of the markers is greatlyincreased. Second, an indication of the position of the disease geneamong the markers may be determined. This allows identification offlanking markers, and thus eventually allows identification of a smallregion in which the disease gene resides.

For the initial linkage analysis, the phenotype and asthma affectionstatus were defined by a patient described above who answered thefollowing questions in the affirmative: (i) have you ever had asthma,(ii) do you have a current physician's diagnosis of asthma, and (iii)are you currently taking asthma medications? Medications include inhaledor oral bronchodilators, cromolyn, theophylline or steroids.

The distribution of the number of genotyped affected siblings was asfollows: 88.7% of the families had 2 siblings, 10.9% had 3 siblings and0.5% had 4 siblings. Ninety eight families were ascertained in the USand 386 in the UK.

Allele sharing methods, implemented in the MAPMAKER/SIBS (Kruglyak L &Lander E S, Am J Hum Genet. 57:439-454, 1995), were used on our sampleof affected sibling pairs. Multipoint linkage analyses were performedusing 54 polymorphic markers spanning a 162 cM region on both arms ofchromosome 12. The map location and distances between markers wereobtained from the genetic maps published by the Marshfield medicalresearch foundation (http://www.marshmed.org/genetics/). Ambiguous orderin the Marshfield map was resolved using the program MULTIMAP (Matise TC et al., Nature Genet. 6:384-390, 1994) on the 46.

FIG. 1 displays the multipoint LOD score against the map location ofmarkers along chromosome 12. A Maximum LOD Score (MLS) of 2.9 wasobtained at location 161.7 cM, 1.0 cM distal to markers D12597 andD1251045. An excess sharing by descent (Identity By Descent, IBD=2) of0.31 was observed at the maximum LOD score. Table 1 lists the single andmultipoint LOD scores at each marker.

These data suggest that chromosome 12 is a location that may contain agene or genes involved in asthma and diseases thereof.

TABLE 1 Chromosome 12 Linkage Analysis Marker Distance Two-pointMultipoint D12S372 6.4 0.0 0.0 GATA49D12 17.7 0.0 0.0 D12S77 20.3 0.00.0 D12S391 26.2 0.0 0.0 D12S358 26.2 0.0 0.0 D12S364 30.6 0.2 0.0D12S373 36.1 0.0 0.0 D12S1042 48.7 0.0 0.0 GATA91H06 56.3 0.0 0.0D12S368 66.0 0.2 0.3 D12S398 68.2 0.2 0.4 D12S83 75.2 1.1 0.0 D12S129478.1 0.0 0.0 IFNgama 80.4 0.0 0.0 D12S375 80.5 0.3 0.0 D12S43 80.5 0.30.0 D12S1052 83.2 0.0 0.0 D12S92 83.2 1.0 0.0 D12S326 86.4 0.1 0.1D12S64 89.4 0.0 0.2 D12S379 93.7 0.0 0.1 D12S311 94.5 0.1 0.0 D12S8295.0 0.1 0.1 D12S819 95.0 0.0 0.1 D12S1064 95.0 0.0 0.0 D12S95 96.1 0.20.2 D12S829 97.2 0.1 0.6 D12S1706 104.1 0.6 0.4 D12S1300 104.1 0.2 0.3D12S1727 107.2 0.0 0.1 D12S1607 107.9 0.0 0.1 IGF1 109.5 0.0 0.0 PAH109.5 0.0 0.0 D12S360 111.3 0.0 0.0 D12S338 111.9 0.0 0.0 D12S78 111.90.0 0.0 D12S811 120.7 0.1 0.3 D12S1341 123.0 0.0 0.5 NOS1 123.1 0.1 0.4D12S2070 125.3 0.2 0.7 D12S366 133.3 1.2 1.7 D12S1619 134.5 0.8 1.8D12S385 135.1 2.0 1.6 PLA2G1B 136.8 0.9 1.4 D12S395 136.8 2.1 1.5D12S300 140.2 0.9 1.7 D12S342 144.8 1.6 2.2 D12S324 147.2 1.3 1.4D12S2078 149.6 0.9 1.9 D12S1659 155.9 0.3 1.6 D12S97 160.7 0.9 2.7D12S1045 160.7 3.0 2.8 D12S392 165.7 1.1 2.3 D12S357 168.8 0.8 1.1

D. Linkage Results

The linkage results for chromosome 12 described above were used todelineate a candidate region for disorder-associated gene(s) located onchromosome 12. Gene discovery efforts were initiated in a ˜43 cMinterval from marker D12S2070 to the 12q telomere, representing a 99%confidence interval. All genes known to map to this interval wereconsidered as candidates. The discovery of novel genes using direct cDNAselection focused on a ˜15 cM region approximately between markersD12S1609 and D12S357.

The following section describes details of the efforts to generatecloned coverage of the disorder gene region on chromosome 12, i.e.,construction of a BAC contig spanning the region. There are two primaryreasons for this: 1) to provide genomic clones for DNA sequencing;analysis of this sequence provides information about the gene content ofthe region, and 2) to provide reagents for direct cDNA selection; thisprovides additional information about novel genes mapping to theinterval. The physical map consists of an ordered set of molecularlandmarks, and a set of bacterial artificial chromosome (BAC, Kim,U.-J., et al., (1996), Genomics 34, 213-218 and Shizuya, H., et al.,(1992). Proc. Natl. Acad. Sci. USA 89, 8794-8797) clones that containthe disorder gene region from chromosome 12q23-qter.

FIG. 2 depicts the STS content of BAC RP11-0702113 in 12q23-qter. Gene214 is located within this BAC as indicated at the top of the figure.Markers used to screen the RPCI-11 BAC library (P. deJong—Roswell ParkCancer Institute) are shown vertically above the solid black horizontalline. The following steps were performed:

1. Map Integration. Various publicly available mapping resources wereutilized to identify existing STS markers (Olson et al, (1989), Science,245:1434-1435) in the 12q23-qter region. Resources included the GenomeDatabase (GDB) at the website of: (hypertext transfer protocol,genomedatabase, world wide web.gdb.org/); Genethon at the website of:(hypertext transfer protocol, world wide web. genethon-en.html);Marshfield Center for Medical Genetics at the website of: (hypertexttransfer protocol, world wide web. marshmed.org/genetics/); theWhitehead Institute Genome Center at the website of: (hypertext transferprotocol, world wide web-genome.wi.mit.edu/); GeneMap98, dbSTS and dbESTat the website of: NCBI, (hypertext transfer protocol, world wide web,ncbi.nlm.nih.gov/); the Sanger Centre at the website of: (hypertexttransfer protocol, world wide web.sanger.ac.uk/); and the Stanford HumanGenome Center at the website of: (hypertext transfer protocol, worldwide web-shgc.stanford.edu/). Maps were integrated manually to identifymarkers mapping to the disorder region. A list of the markers isprovided in Table 2.

2. Marker Development. Sequences for existing STSs were obtained fromthe GDB, RHDB at the website of the Genome Database, RHDB, (hypertexttransfer protocol, world wide web. ebi.ac.uk/RHdb/), or NCBI and wereused to pick primer pairs (overgos, See Table 2) for BAC libraryscreening. Novel markers were developed either from publicly availablegenomic sequences, proprietary cDNA sequences or from sequences derivedfrom BAC insert ends (described below). Primers were chosen using ascript that automatically performs vector and repetitive sequencemasking using Crossmatch (P. Green, U. of Washington); subsequent primerpicking was performed using a customized Filemaker Pro database. Primersfor use in PCR-based clone confirmation or radiation hybrid mapping(described below) were chosen using the program Primer3 (Steve Rozen,Helen J. Skaletsky (1996, 1997); Primer3 is available at the website of(hypertext transfer protocol, world wideweb,—genome.wi.mit.edu/genomesoftware;other/primer3.htm/).

TABLE 2 DNA Forward Reverse Overgo Locus Type Gene Primer PrimerB0702C13A1x BACend GTAGTAACA AGAGAGGAA GAATGGACT CAGCATCAA TTGA AGTC(SEQ ID (SEQ ID NO: 12) NO: 13) A005Q05 EST CAAACAGGG GTGTTTCAGTCCACCGTG CCACATTTC GAAA CACG (SEQ ID (SEQ ID NO: 14) NO: 15) Th GeneMucin ATCCACCGC GACCATCAA 8 TAGAAACCC CTGATGAGT (MUC8) ACTC GGGT (SEQ ID(SEQ ID NO: 16) NO: 17) B0702C13A1y BACend TCATGGGGG TGGCCTCAA TGCTTTGACAGGCTCAAG CTTG GTCA (SEQ ID (SEQ ID NO: 18) NO: 19)

3. Radiation Hybrid (RH) Mapping. Radiation hybrid mapping was performedagainst the Genebridge4 panel (Gyapay, et al., (1996), Hum. Mol. Genet.5:339-46) purchased from Research Genetics, in order to refine thechromosomal localization of genetic markers used in genotyping as wellas to identify, confirm and refine localizations of markers fromproprietary sequences. Standard PCR procedures were used for typing theRH panel with markers of interest. Briefly, 10 μl PCR reactionscontained 25 ng DNA of each of the 93 Genebridge4 RH samples. PCRproducts were electrophoresed in 2% agarose gels (Sigma) containing 0.5μg/ml ethidium bromide in 1×TBE at 150 volts for 45 min. Theelectrophoresis units used were the Model A3-1 systems from OwlScientific Products. Typically, gels contained 10 tiers of lanes with 50wells/tier. Molecular weight markers (100 bp ladder, GIBCO/BRL) wereloaded at both ends of the gel. Images of the gels were captured with aKodak DC40 CCD camera and processed with Kodak 1D software. The gel datawere exported as tab delimited text files; names of the files includedinformation about the panel screened, the gel image files and the markerscreened. These data were automatically imported using a customized Perlscript into Filemaker databases for data storage and analysis. The datawere then automatically formatted and submitted to an internal serverfor linkage analysis to create a radiation hybrid map using RHMAPPER(Stein, L., Kruglyak, L., Slonim, D., and El Lander (1995); availablefrom the Whitehead Institute/MIT Center for Genome Research, athttp://www.genome.wi.mit.edu/ftp/pub/software/rhmapper/, and viaanonymous ftp to ftp.genome.wi.mit.edu, in thedirectory/pub/software/rhmapper.) The RH mapping results obtained forGene 214 indicate that it is present in the 12Q 12-qter region at 507.12cRays on the 684 coordinate system.

4. BAC Library Screening. The protocol used for BAC library screeningwas based on the “overgo” method, originally developed by John McPhersonat Washington University in St. Louis(http://www.tree.caltech.edu/protocols/overgo.html, and Cai, W—W., etal., (1998), Genomics 54:387-397). This method involves filling in theoverhangs generated after annealing two primers, each 22 nucleotides inlength, that overlap by 8 nucleotides. The resulting labeled 36 bpproduct is then used in hybridization-based screening of high densitygrids derived from the RPCI-11 BAC library (Pieter deJong, Roswell ParkCancer Institute, http://bacpac.med.buffalo.edu). Typically, 15 probeswere pooled together in one hybridization of 12 filters (13.5 genomeequivalents).

Stock solutions (2 μM) of combined complementary oligos were heated at80° C. for 5 min, then placed at 37° C. for 10 min followed by storageon ice. Labeling reactions were set up as follows: 1.0 μl H₂O, 5 μlmixed oligos—2 μM each, 0.5 μl BSA (2 mg/ml), 2 μl OLB(-A, -C, -N6)Solution (see below), 0.5 μl ³²P-dATP (3000 Ci/mmol), 0.5 μl ³²P-dCTP(3000 Ci/mmol), 0.5 μl Klenow fragment (5U/μl). The reaction wasincubated at room temperature for 1 hr followed by removal ofunincorporated nucleotides with Sephadex G50 spin columns.

OLB(-A, -C, -N6) Solution

Solution O—1.25 M Tris-HCL, pH 8, 125 M MgCl₂

Solution A—1 ml Solution O, 18 μl 2-mercaptoethanol, 5 μl 0.1M dTTP, 5μl 0.1M dGTP

Solution B—2M HEPES-NaOH, pH 6.6 Solution C—3 mM Tris-HCl, pH 7.4, 0.2mM EDTA

Solutions A, B, and C were combined to a final ratio of 1:2.5:1.5,aliquots were stored at −20° C.

High density BAC library membranes were pre-wetted in 2×SSC at 58° C.Filters were then drained slightly and placed in hybridization solution(1% Bovine serum albumin, 1 mM EDTA—pH 8.0, 7% SDS, and 0.5 M sodiumphosphate) pre-warmed to 58° C. and incubated at 58° C. for 2-4 hr.Typically, 6 filters were hybridized per container. Ten ml ofpre-hybridization solution were removed, combined with the denaturedovergo probes, and added back to the filters. Hybridization wasperformed overnight at 58° C. The hybridization solution was removed andfilters were washed once in 2×SSC, 0.1% SDS, followed by a 30 minutewash in the same solution but at 58° C. Filters were then washed in1.5×SSC, 01.% SDS at 58° C. for 30 min. 0.5×SSC, 0.1% SDS at 58° C. for30 min and finally in 0.1×SSC, 0.1% SDS at 58° C. for 30 min. Filterswere then wrapped in Saran Wrap and exposed to film overnight. To removebound probe, filters were treated in 0.1×SSC, 0.1% SDS pre-warmed to 95°C. and allowed to return to room temperature. Clone addresses weredetermined as described by instructions supplied by RPCI.

Recovery of clonal BAC cultures from the library involved streaking outa sample from the appropriate library well onto LB agar (Maniatis, T.,Fritsch, E. F., and J. Sambrook, (1982) Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.)containing 12.5 μg/mlchloramphenicol (Sigma) and incubating overnight. Asingle colony and a portion of the initial streak quadrant wereinoculated into 400 μl LB plus chloramphenicol in wells of a 96 wellplate. Cultures were grown overnight at 37° C. For storage, 100 μl of80% glycerol was added and the plates placed at −80° C. To determine themarker content of clones, aliquots of the 96 well plate cultures weretransferred to the surface of nylon filters (GeneScreen Plus, NEN)placed on LB/chloramphenicol Petri plates. Colonies were grown overnightat 37° C. and colony lysis was performed as follows: Filters were placedon pools of 10% SDS for 3 min, 0.5 N NaOH, 1.5 M NaCl for 5 min, and 0.5M Tris-HCl, pH 7.5, 1 M NaCl for 5 min. Filters were then air dried andwashed free of debris in 2×SSC for 1 hr. The filters were air dried forat least 1 hr and DNA crosslinked linked to the membrane using standardconditions. Probe hybridization and filter washing were performed asdescribed above for the primary library screening. Confirmed clones werestored in LB containing 15% glycerol.

In some cases polymerase chain reaction (PCR) was used to confirm themarker content of clones. PCR conditions for each primer pair wereinitially optimized with respect to MgCl₂ concentration. The standardbuffer was 10 mM Tris-HCl (pH 8.3), 50 mM KCl, MgCl₂, 0.2 mM each dNTP,0.2 μM each primer, 2.7 ng/μl human DNA, 0.25 units of AmpliTaq (PerkinElmer) and MgCl₂ concentrations of 1.0 mM, 1.5 mM, 2.0 mM or 2.4 mM.Cycling conditions included an initial denaturation at 94° C. for 2minutes followed by 40 cycles at 94° C. for 15 seconds, 55° C. for 25seconds, and 72° C. for 25 seconds followed by a final extension at 72°C. for 3 minutes. Depending on the results from the initial round ofoptimization the conditions were further optimized if necessary.Variables included increasing the annealing temperature to 58° C. or 60°C., increasing the cycle number to 42 and the annealing and extensiontimes to 30 seconds, and using AmpliTaqGold (Perkin Elmer).

5. BAC DNA Preparation. Several different types of DNA preparationmethods were used for isolation of BAC DNA. The manual alkaline lysisminiprep protocol listed below (Maniatis, T., Fritsch, E. F., and J.Sambrook, (1982) Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.) was successfully used formost applications, i.e., restriction mapping, CHEF gel analysis and FISHmapping, but was not reproducibly successful in endsequencing. TheAutogen protocol described below was used specifically for BAC DNApreparation for endsequencing purposes.

For manual alkaline lysis BAC minipreps, bacteria were grown in 15 mlTerrific Broth containing 12.5 μg/ml chloramphenicol in a 50 ml conicaltube at 37° C. for 20 hrs with shaking at 300 rpm. The cultures werecentrifuged in a Sorvall RT 6000 D at 3000 rpm (1800×g) at 4° C. for 15min. The supernatant was then aspirated as completely as possible. Insome cases cell pellets were frozen at −20° C. at this step for up to 2weeks. The pellet was then vortexed to homogenize the cells and minimizeclumping. 250 μl of P1 solution (50 mM glucose, 15 mM Tris-HCl, pH 8, 10mM EDTA, and 100 μg/ml RNase A) was added and the mixture pipeted up anddown to mix. The mixture was then transferred to a 2 ml Eppendorf tube.350 μl of P2 solution (0.2 N NaOH, 1% SDS) was then added, and themixture mixed gently and incubated for 5 min at room temperature. 350 μlof P3 solution (3M KOAc, pH 5.5) was added and the mixture mixed gentlyuntil a white precipitate formed. The solution was incubated on ice for5 min and then centrifuged at 4° C. in a microfuge for 10 min. Thesupernatant was transferred carefully (avoiding the white precipitate)to a fresh 2 ml Eppendorf tube, and 0.9 ml of isopropanol was added; thesolution was mixed and left on ice for 5 min. The samples werecentrifuged for 10 min, and the supernatant removed carefully. Pelletswere washed in 70% ethanol and air dried for 5 min. Pellets wereresuspended in 200 μl of TE8 (10 mM Tris-HCl, pH 8.0, 1.0 mM EDTA, pH8.0), and RNase (Boehringer Mannheim) added to 100 μg/ml. Samples wereincubated at 37° C. for 30 min, then precipitated by addition of NH₄OActo 0.5 M and 2 volumes of ethanol. Samples were centrifuged for 10 min,and the pellets washed with 70% ethanol followed by air drying anddissolving in 50 μl TE8. Typical yields for this DNA prep were 3-5 μg/15ml bacterial culture. Ten to 15 μl were used for EcoRI restrictionanalysis; 5 μl was used for NotI digestion and clone insert sizing byCHEF gel electrophoresis.

Autogen 740 BAC DNA preparations for endsequencing were prepared bydispensing 3 ml of LB media containing 12.5 μg/ml of chloramphenicolinto autoclaved Autogen tubes. A single tube was used for each clone.For inoculation, glycerol stocks were removed from −70° C. storage andplaced on dry ice. A small portion of the glycerol stock was removedfrom the original tube with a sterile toothpick and transferred into theAutogen tube; the toothpick was left in the Autogen tube for at leasttwo minutes before discarding. After inoculation the tubes were coveredwith tape making sure the seal was tight. When all samples wereinoculated, the tube units were transferred into an Autogen rack holderand placed into a rotary shaker at 37° C. for 16-17 hours at 250 rpm.Following growth, standard conditions for BAC DNA preparation, asdefined by the manufacturer, were used to program the Autogen. Sampleswere not dissolved in TE8 as part of the program—DNA pellets were leftdry. When the program was complete the tubes were removed from theoutput tray and 30 μl of sterile distilled and deionized H2O was addeddirectly to the bottom of the tube. The tubes were then gently shakenfor 2-5 seconds and then covered with parafilm and incubated at roomtemperature for 1-3 hours. DNA samples were then transferred to anEppendorf tube and used either directly for sequencing or stored at 4°C. for later use.

6. BAC Clone Characterization. DNA samples prepared either by manualalkaline lysis or the Autogen protocol were digested with EcoRI foranalysis of restriction fragment sizes. These data were used to comparethe extent of overlap among clones. Typically 1-2 μg were used for eachreaction. Reaction mixtures included: 1× Buffer 2 (New England Biolabs),0.1 mg/ml bovine serum albumin (New England Biolabs), 50 μg/ml RNase A(Boehringer Mannheim), and 20 units of EcoRI (New England Biolabs) in afinal volume of 25 μl. Digestions were incubated at 37° C. for 4-6hours. BAC DNA was also digested with NotI for estimation of insert sizeby CHEF gel analysis (see below). Reaction conditions were identical tothose for EcoRI except that 20 units of NotI were used. Six μl of 6×Ficoll loading buffer containing bromphenol blue and xylene cyanol wasadded prior to electrophoresis.

EcoRI digests were analyzed on 0.6% agarose (Seakem, FMC Bioproducts) in1×TBE containing 0.5 μg/ml ethidium bromide. Gels (20 cm×25 cm) wereelectrophoresed in a Model A4 electrophoresis unit (Owl Scientific) at50 volts for 20-24 hrs. Molecular weight size markers includedundigested lambda DNA, HindIII digested lambda DNA, and HaeIII digested.X174 DNA. Molecular weight markers were heated at 65° C. for 2 minprior to loading the gel. Images were captured with a Kodak DC40 CCDcamera and analyzed with Kodak 1D software.

NotI digests were analyzed on a CHEF DRII (BioRad) electrophoresis unitaccording to the manufacturer's recommendations. Briefly, 1% agarosegels (BioRad pulsed field grade) were prepared in 0.5×TBE, equilibratedfor 30 min in the electrophoresis unit at 14° C., and electrophoresed at6 volts/cm for 14 hrs with circulation. Switching times were ramped from10 sec to 20 sec. Gels were stained after electrophoresis in 0.5 μg/mlethidium bromide. Molecular weight markers included undigested lambdaDNA, HindIII digested lambda DNA, lambda ladder PFG ladder, and lowrange PFG marker (all from New England Biolabs).

7. BAC Endsequencing. The sequence of BAC insert ends utilized DNAprepared by either of the two methods described above. The ends of BACclones were sequenced for the purpose of filling gaps in the physicalmap and for gene discovery information. The following vector primersspecific to the BAC vector pBACe3.6 were used to generate endsequencefrom BAC clones:

pBAC 5'-2 TGT AGG ACT ATA TTG CTC (SEQ ID NO: 20) andpBAC 3'-1 CGA CAT TTA GGT GAC ACT. (SEQ ID NO: 21)

The following sequencing protocol using ABI dye-terminator chemistry wasused to set up sequencing reactions for 96 clones. The BigDye (Mix:Perkin Elmer/ABI BigDye) Terminator Ready Reaction Mix with AmpliTaq”FS, Part number 4303151, was used for sequencing with fluorescentlylabelled dideoxy nucleotides. A master sequencing mix was prepared foreach primer reaction set including:

1600 μl of BigDye terminator mix (ABI)800 μl of 5×CSA buffer (ABI)800 μl of primer (either pBAC 5′-2 or pBAC 3′-1 at 3.2 μM)

The sequencing cocktail was vortexed to ensure it was well-mixed and 32μl was aliquoted into each PCR tube. Eight μl of the Autogen DNA foreach clone was transferred from the DNA source plate to a correspondingwell of the PCR plate. The PCR plates were sealed tightly andcentrifuged briefly to collect all the reagents. Cycling conditions wereas follows:

95° C. for 5 minutes

95° C. for 30 seconds

50° C. for 20 seconds

65° C. for 4 minutes

Go to steps 2 through 4 above for an additional 74 times

4° C. forever

At the end of the sequencing reaction, the plates were removed from thethermocycler and centrifuged briefly. Centri·Sep 96 plates were thenused according to manufacturer's recommendation to remove unincorporatednucleotides, salts and excess primers. Each sample was resuspended in1.5 μl of loading dye of which 1.3 μl was loaded on ABI 377 FluorescentSequencers. The resulting endsequences were then used to develop markersto rescreen the BAC library for filling gaps and were also analyzed byBLAST searching for EST or gene content.

E. Sub-Cloning and Sequencing of BACS from 12q23-qter

The physical map of the chromosome 12 region provides the BAC clone andlocation for use as sequencing templates (see FIG. 2). DNA sequencing ofthe BAC RPCI-11_(—)0702C13 from the region is contained within (SEQ IDNO: 1).

DNA for BAC RPCI-11_(—)0702C13 (the “BAC DNA”) was isolated according toone of two protocols: either a Qiagen purification of BAC DNA (Qiagen,Inc., Chatsworth, Calif., per manufacturer's instructions) or a manualpurification using a method which is a modification of the standardalkaline lysis/Cesium Chloride preparation of plasmid DNA (see e.g.,Ausubel et al, (1997), Current Protocols in Molecular Biology, JohnWiley & Sons). Briefly, for the manual protocol, cells were pelleted,resuspended in GTE (50 mM glucose, 25 mM Tris-Cl (pH 8), 10 mM EDTA) andlysozyme (50 mg/ml solution), followed by NaOH/SDS (1% SDS/0.2N NaOH)and then an ice-cold solution of 3M KOAc (pH 4.5-4.8). RNaseA was addedto the filtered supernatant, followed by treatment with Proteinase K and20% SDS. The DNA was then precipitated with isopropanol, dried andresuspended in TE (10 mM Tris, 1 mM EDTA (pH 8.0)). The BAC DNA wasfurther purified by Cesium Chloride density gradient centrifugation(Ausubel et al, (1997), Current Protocols in Molecular Biology, JohnWiley & Sons).

Following isolation, the BAC DNA was hydrodynamically sheared using HPLC(Hengen, et al., (1997), Trends in Biochem. Sci., 22:273-274) to aninsert size of 2000-3000 bp. After shearing, the DNA was concentratedand separated on a standard 1% agarose gel. A single fraction,corresponding to the approximate size, was excised from the gel andpurified by electroelution (Sambrook et al, (1989), Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring, N.Y.).

The purified DNA fragments were then blunt-ended using T4 DNApolymerase. The healed DNA was then ligated to unique BstXI-linkeradapters (5′ GTCTTCACCACGGGG (SEQ ID NO: 22) and 5′ GTGGTGAAGAC (SEQ IDNO: 23) in 100-1000 fold molar excess). These linkers are complimentaryto the BstXI-cut pMPX vectors, while the overhang is notself-complimentary. Therefore, the linkers will not concatemerize norwill the cut-vector re-ligate to itself easily. The linker-adaptedinserts were separated from unincorporated linkers on a 1% agarose geland purified using GeneClean (BIO 101, Inc.). The linker-adapted insertwas then ligated to a modified pBlueScript vector to construct a“shotgun” subclone library. The vector contains an out-of-frame lacZgene at the cloning site which becomes in-frame in the event that anadapter-dimer is cloned, allowing these to be avoided by their bluecolor.

All subsequent steps were based on sequencing by ABI377 automated DNAsequencing methods. Only major modifications to the protocols arehighlighted. Briefly, the library was then transformed intoDH5-competent cells (Gibco/BRL, DH5-transformation protocol). Qualitywas assessed by plating onto antibiotic plates containing ampicillin andIPTG/Xgal. The plates were incubated overnight at 37° C. Successfultransformants were then used for plating of clones and picking forsequencing. The cultures were grown overnight at 37° C. DNA was purifiedusing a silica bead DNA preparation (Ng et al, Nucl. Acids Res.,24:5045-5047 (1996)) method. In this manner, 25 μg of DNA was obtainedper clone.

These purified DNA samples were then sequenced using ABI dye-terminatorchemistry. The ABI dye terminator sequence reads were run on ABI377machines and the data were directly transferred to UNIX machinesfollowing lane tracking of the gels. All reads were assembled usingPHRAP (P. Green, Abstracts of DOE Human Genome ProgramContractor-Grantee Workshop V, January 1996, p. 157) with defaultparameters and quality scores. SEQ ID NO:1 comprises a portion of theBAC which includes the genomic sequence of Gene 214

F. Gene Identification

Any gene or EST mapping to the interval based on public map data orproprietary map data was considered a candidate disorder gene.

1. Gene Identification from clustered DNA fragments. DNA sequencescorresponding to gene fragments in public databases (Genbank and humandbEST) and proprietary cDNA sequences (IMAGE consortium and directselected cDNAs) were masked for repetitive sequences and clustered usingthe PANGEA Systems (Oakland, Calif.) EST clustering tool. The clusteredsequences were then subjected to computational analysis to identifyregions bearing similarity to known genes. This protocol included thefollowing steps:

-   -   i. The clustered sequences were compared to the publicly        available Unigene database (National Center for Biotechnology        Information, National Library of Medicine, 38A, 8N905, 8600        Rockville Pike, Bethesda, Md. 20894; www.ncbi.nlm.nih.gov) using        the blastn2 algorithm (Altschul et al, Nucl. Acids Res.,        25:3389-3402 (1997)). The parameters for this search were:        E=0.05, v=50, B=50 (where E is the expected probability score        cutoff, V is the number of database entries returned in the        reporting of the results, and B is the number of sequence        alignments returned in the reporting of the results (Altschul et        al, J. Mol. Biol., 215:403-410 (1990)).    -   ii. The clustered sequences were compared to the Genbank        database (National Center for Biotechnology Information,        National Library of Medicine, 38A, 8N905, 8600 Rockville Pike,        Bethesda, Md. 20894; www.ncbi.nlm.nih.gov) using blastn2        (Altschul et al, Nucl. Acids. Res., 25:3389-3402 (1997)). The        parameters for this search were E=0.05, V=50, B=50, where E, V,        and B are defined as above.    -   iii. The clustered sequences were translated into protein for        all six reading frames, and the protein sequences were compared        to a non-redundant protein database compiled from Genpept        Swissprot PIR (National Center for Biotechnology Information,        National Library of Medicine, 38A, 8N905, 8600 Rockville Pike,        Bethesda, Md. 20894; www.ncbi.nlm.nih.gov). The parameters for        this search were E=0.05, V=50, B=50, where E, V, and B are        defined as above.    -   iv. The clustered sequences were compared to BAC sequences (see        below) using blastn2 (Altschul et al, Nucl. Acids. Res.,        25:3389-3402 (1997)). The parameters for this search were        E=0.05, V=50, B=50, where E, V, and B are defined as above.

2. Gene Identification from BAC Genomic Sequence. Following assembly ofthe BAC sequences into contigs, the contigs were subjected tocomputational analyses to identify coding regions and regions bearingDNA sequence similarity to known genes. This protocol included thefollowing steps:

-   -   i. Contigs were degapped. The sequence contigs often contain        symbols (denoted by a period symbol) that represent locations        where the individual ABI sequence reads have insertions or        deletions. Prior to automated computational analysis of the        contigs, the periods were removed. The original data were        maintained for future reference.    -   ii. BAC vector sequences were “masked” within the sequence by        using the program crossmatch (Phil Green,        http:\\chimera.biotech.washington.edu\UWGC). Since the shotgun        library construction detailed above left some BAC vector in the        shotgun libraries, this program was used to compare the sequence        of the BAC contigs to the BAC vector and to mask any vector        sequence prior to subsequent steps. Masked sequence was marked        by an “X” in the sequence files, and remained inert during        subsequent analyses.    -   iii. E. coli sequences contaminating the BAC sequences were        masked by comparing the BAC contigs to the entire E. coli DNA        sequence.    -   iv. Repetitive elements known to be common in the human genome        were masked using crossmatch. In this implementation of        crossmatch, the BAC sequence is compared to a database of human        repetitive elements (Jerzy Jerka, Genetic Information Research        Institute, Palo Alto, Calif.). The masked repeats were marked by        X and remained inert during subsequent analyses.    -   v. The location of exons within the sequence was predicted using        the MZEF computer program (Zhang, Proc. Natl. Acad. Sci.,        94:565-568 (1997); GenScan (Burge and Karlin, J. Mol. Biol.,        268:78-94)).        vi. The sequence was compared to the publicly available unigene        database (National Center for Biotechnology Information,        National Library of Medicine, 38A, 8N905, 8600 Rockville Pike,        Bethesda, Md. 20894; www.ncbi.nlm.nih.gov) using the blastn2        algorithm (Altschul et al, Nucl. Acids Res., 25:3389-3402        (1997)). The parameters for this search were: E=0.05, v=50, B=50        (where E is the expected probability score cutoff, V is the        number of database entries returned in the reporting of the        results, and B is the number of sequence alignments returned in        the reporting of the results (Altschul et al, J. Mol. Biol.,        215:403-410 (1990)).    -   vii. The sequence was translated into protein for all six        reading frames, and the protein sequences were compared to a        non-redundant protein database compiled from Genpept Swissprot        PIR (National Center for Biotchnology Information, National        Library of Medicine, 38A, 8N905, 8600 Rockville Pike, Bethesda,        Md. 20894; www.ncbi.nlm.nih.gov). The parameters for this search        were E=0.05, V=50, B=50, where E, V, and B are defined as above.    -   viii. The BAC DNA sequence was compared to a database of        clustered sequences using blastn2 (Altschul et al, Nucl. Acids.        Res., 25:3389-3402 (1997)). The parameters for this search were        E=0.05, V=50, B=50, where E, V, and B are defined as above. The        database of clustered sequences was prepared utilizing a        proprietary clustering technology (Pangea Systems, Inc.) using        cDNA clones derived from direct selection experiments (described        below), human dbEST mapping to the 12q23-qter region,        proprietary cDNAs, Genbank genes and IMAGE consortium cDNA        clones.    -   ix. The BAC sequence was compared to the sequences derived from        the ends of BACs from the region on chromosome 12 using blastn2        (Altschul et al, Nucl. Acids. Res., 25:3389-3402 (1997)). The        parameters for this search were E=0.05, V=50, B=50, where E, V,        and B are defined as above.    -   x. The BAC sequence was compared to the Genbank database        (National Center for Biotechnology Information, National Library        of Medicine, 38A, 8N905, 8600 Rockville Pike, Bethesda, Md.        20894; www.ncbi.nlm.nih.gov) using blastn2 (Altschul et al,        Nucl. Acids. Res., 25:3389-3402 (1997)). The parameters for this        search were E=0.05, V=50, B=50, where E, V, and B are defined as        above.    -   xi. The BAC sequence was compared to the STS division of Genbank        database (National Center for Biotechnology Information,        National Library of Medicine, 38A, 8N905, 8600 Rockville Pike,        Bethesda, Md. 20894; www.ncbi.nlm.nih.gov) using blastn2        (Altschul et al., 1997). The parameters for this search were        E=0.05, V=50, B=50, where E, V, and B are defined as above.    -   xii. The BAC sequence was compared to the Expressed Sequence Tag        (EST) Genbank database (National Center for Biotchnology        Information, National Library of Medicine, 38A, 8N905, 8600        Rockville Pike, Bethesda, Md. 20894; www.ncbi.nlm.nih.gov) using        blastn2 (Altschul et al., Nucl. Acids. Res., 25:3389-3402        (1997)). The parameters for this search were E=0.05, V=50, B=50,        where E, V, and B are defined as above.    -   xiii. The BAC sequence was compared to the Expressed Sequence        Tag (EST) Genbank database (National Center for Biotchnology        Information, National Library of Medicine, 38A, 8N905, 8600        Rockville Pike, Bethesda, Md. 20894; www.ncbi.nlm.nih.gov) using        blastn2 (Altschul et al., Nucl. Acids. Res., 25:3389-3402        (1997)). The parameters for this search were E=0.05, V=50, B=50,        where E, V, and B are defined as above.        G. cDNA Cloning and Expression Analysis

1. Construction of cDNA libraries. Directionally cloned cDNA librariesfrom normal lung and bronchial epithelium were constructed usingstandard methods described previously (Soares et. al., 1994, AutomatedDNA Sequencing and Analysis, Adams, Fields and Venter, Eds., AcademicPress, NY, pages 110-114). Total and cytoplasmic RNAs were extractedfrom tissue or cells by homogenizing the sample in the presence ofGuanidinium Thiocyanate-Phenol-Chloroform extraction buffer (e.g.Chomczynski and Sacchi, Anal. Biochem., 162:156-159 (1987)) using apolytron homogenizer (Brinkman Instruments). PolyA+ RNA was isolatedfrom total/cytoplasmic RNA using dynabeads-dT according to themanufacturer's recommendations (Dynal, Inc.). The ds cDNA synthesizedwas then ligated into the plasmid vector pBluescript II KS+ (Stratagene,La Jolla, Calif.), and the ligation mixture was transformed into E. colihost DH10B or DH12S by electroporation (Soares, 1994). Followingovernight growth at 37° C., DNA was recovered from the E. coli coloniesafter scraping the plates by processing as directed for the Mega-prepkit (Qiagen, Chatsworth, Calif.). The quality of the cDNA libraries wasestimated by counting a portion of the total number of primarytransformants, determining the average insert size and the percentage ofplasmids with no cDNA insert. Additional cDNA libraries (human totalbrain, heart, kidney, leukocyte, and fetal brain) were purchased fromLife Technologies, Bethesda, Md.

cDNA libraries, both oligo (dT) and random hexamer-primed were used forisolating cDNA clones mapping within the disorder critical region. Four10×10 arrays of each of the cDNA libraries were prepared as follows: thecDNA libraries were titered to 2.5×10⁶ using primary transformants. Theappropriate volume of frozen stock was used to inoculate 2 L ofLB/ampicillin (100 μg/μl). 400 aliquots containing 4 ml of theinoculated liquid culture were generated. Each tube contained about 5000cfu. The tubes were incubated at 30° C. overnight with shaking until anOD of 0.7-0.9 was obtained. Frozen stocks were prepared for each of thecultures by aliquotting 300 μl of culture and 100 μl of 80% glycerol.Stocks were frozen in a dry ice/ethanol bath and stored at −70° C. DNAwas isolated from the remaining culture using the Qiagen (Chatsworth,Calif.) spin mini-prep it according to the manufacturer's instructions.The DNAs from the 400 cultures were pooled to make 80 column and rowpools. Markers were designed to amplify putative exons from candidategenes. Once a standard PCR condition was identified and specific cDNAlibraries were determined to contain cDNA clones of interest, themarkers were used to screen the arrayed library. Positive addressesindicating the presence of cDNA clones were confirmed by a second PCRusing the same markers.

Once a cDNA library was identified as likely to contain cDNA clonescorresponding to a specific transcript of Gene 214, it was used toisolate a clone or clones containing cDNA inserts. This was accomplishedby a modification of the standard “colony screening” method (Sambrook etal, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor N.Y. (1989)). Specifically, twenty 150 mmLB+ampicillin agar plates were spread with 20,000 colony forming units(cfu) of cDNA library and the colonies allowed to grow overnight at 37°C. Colonies were transferred to nylon filters (Hybond from Amersham, orequivalent) and duplicates prepared by pressing two filters togetheressentially as described (Sambrook et al, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring HarborN.Y. (1989)). The “master” plate was then incubated an additional 6-8hrs to allow the colonies additional growth. The DNA from the bacterialcolonies was then bound onto the nylon filters by treating the filterssequentially with denaturing solution (0.5 N NaOH, 1.5 M NaCl) for twominutes, neutralization solution (0.5 M Tris-Cl pH 8.0, 1.5 M NaCl) fortwo minutes (twice). The bacterial colonies were removed from thefilters by washing in a solution of 2×SSC/2% SDS for one minute whilerubbing with tissue paper. The filters were air dried and baked undervacuum at 80° C. for 1-2 hrs to cross link the DNA to the filters.

cDNA hybridization probes were prepared by random hexamer labelling(Fineberg and Vogelstein, Anal. Biochem., 132:6-13 (1983)) or byincluding gene-specific primers and no random hexamers in the reaction(for small fragments). The colony membranes were then pre-washed in 10mM Tris-Cl pH 8.0, 1 M NaCl, 1 mM EDTA, 0.1% SDS for 30 minutes at 55°C. Following the pre-wash, the filters were pre-hybridized in >2ml/filter of 6×SSC, 50% deionized formamide, 2% SDS, 5×Denhardt'ssolution, and 100 mg/ml denatured salmon sperm DNA, at 42° C. for 30minutes. The filters were then transferred to hybridization solution(6×SSC, 2% SDS, 5×Denhardt's, 100 mg/ml denatured salmon sperm DNA)containing denatured a-32P-dCTP-labelled cDNA probe and incubatedovernight at 42° C.

The following morning, the filters were washed under constant agitationin 2×SSC, 2% SDS at room temperature for 20 minutes, followed by twowashes at 65° C. for 15 minutes each. A second wash was performed in0.5×SSC, 0.5% SDS for 15 minutes at 65° C. Filters were then wrapped inplastic wrap and exposed to radiographic film. Individual colonies onplates were aligned with the autoradiograph and positive clones pickedinto a 1 ml solution of LB Broth containing ampicillin. After shaking at37° C. for 1-2 hours, aliquots of the solution were plated on 150 mmplates for secondary screening. Secondary screening was identical toprimary screening (above) except that it was performed on platescontaining ˜250 colonies so that individual colonies could be clearlyidentified. Positive cDNA clones were characterized by restrictionendonuclease cleavage, PCR, and direct sequencing to confirm thesequence identity between the original probe and the isolated clone.

To obtain the full-length cDNA, novel sequence from the 5′-end of theclone was used to reprobe the library. This process is repeated untilthe length of the cDNA cloned matched that of the mRNA, estimated byNorthern analysis.

Rapid Amplification of cDNA ends (RACE) was performed following themanufacturer's instructions using a Marathon cDNA Amplification Kit(Clontech, Palo Alto, Calif.) as a method for cloning the 5′ and 3′ endsof candidate genes. cDNA pools were prepared from total RNA byperforming first strand synthesis, where a sample of total RNA samplewas mixed with a modified oligo (dT) primer, heated to 70° C., cooled onice and followed by the addition of: 5× first strand buffer, 10 mM dNTPmix, and AMV Reverse Transcriptase (20 U/μ1). The reaction mixture wasincubated at 42° C. for an hour and placed on ice. For second strandsynthesis, the following components were added directly to the reactiontube: 5× second strand buffer, 10 mM dNTP mix, sterile water, 20× secondstrand enzyme cocktail and the reaction tube was incubated at 16° C. for1.5 hours. T4 DNA Polymerase was added to the reaction tube andincubated at 16° C. for 45 minutes. The second-strand synthesis wasterminated with the addition of an EDTA/Glycogen mix. The sample wassubjected to a phenol/chloroform extraction and an ammonium acetateprecipitation. The cDNA pools were checked for quality by analyzing onan agarose gel for size distribution. Marathon cDNA adapters were thenligated onto the cDNA ends. The specific adapters contained primingsites that allowed for amplification of either 5′ or 3′ ends, and varieddepending on the orientation of the gene specific primer (GSP) that waschosen. An aliquot of the double stranded cDNA was added to thefollowing reagents: 10 μM Marathon cDNA adapter, 5×DNA ligation buffer,T4 DNA ligase. The reaction was incubated at 16° C. overnight and heatinactivated to terminate the reaction. PCR was performed by the additionof the following to the diluted double stranded cDNA pool: 10×cDNA PCRreaction buffer, 10 μM dNTP mix, 10 μM GSP, 10 μM AP1 primer (kit), 50×Advantage cDNA Polymerase Mix. Thermal Cycling conditions were 94° C.for 30 seconds, 5 cycles of 94° C. for 5 seconds, 72° C. for 4 minutes,5 cycles of 94° C. for 5 seconds, 70° C. for 4 minutes, 23 cycles of 94°C. for 5 seconds, 68° C. for 4 minutes. After the first round of PCR wasperformed using the GSP to extend to the end of the adapter to createthe adapter primer binding site, exponential amplification of thespecific cDNA of interest was performed. Usually, a second, nested PCRwas performed to provide specificity. The RACE product was analyzed onan agarose gel. Following excision from the gel and purification(GeneClean, BIO 101), the RACE product was then cloned into pCTNR(General Contractor DNA Cloning System, 5′-3′, Inc.) and sequenced toverify that the clone was specific to Gene 214.

2. Expression Analysis. To characterize the expression of Gene 214, aseries of experiments were performed. First, oligonucleotide primerswere designed for use in the polymerase chain reaction (PCR) so thatportions of a cDNA, EST, or genomic DNA could be amplified from a poolof DNA molecules or RNA population (RT-PCR). The PCR primers were usedin a reaction containing genomic DNA to verify that they generated aproduct of the predicted size (based on the genomic sequence). Acritical piece of data that is required when characterizing novel genesis the length, in nucleotides, of the processed transcript or messengerRNA (mRNA). Those skilled in the art primarily determine the length ofan mRNA by Northern analysis (Sambrook et al, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring HarborN.Y. (1989)). Probes were generated using one of the methods describedbelow. Briefly, sequence verified IMAGE consortium cDNA clones weredigested with appropriate restriction endonucleases to release theinsert. The restriction digest was electrophoresed on an agarose gel andthe bands containing the insert were excised. The gel piece containingthe DNA insert was placed in a Spin-X (Corning Costar Corporation,Cambridge, Mass.) or Supelco spin column (Supelco Park, PA) and spun athigh speed for 15 mins. The DNA was ethanol precipitated and resuspendedin TE. Alternatively, PCR products obtained from genomic DNA or RT-PCRwere also purified as described above. Inserts purified from IMAGEclones were random primer labelled (Feinberg and Vogelstein) to generateprobes for hybridization. Probes from purified PCR products weregenerated by incorporation of a-³²P-dCTP in second round of PCR. FIG. 9is the Northern blot for Gene 214 which includes PolyA+ selected RNAfrom 1) a lymphoblast cell line from as asthmatic individual, 2) lungand 3) trachea. Expression of Gene 214 was detected in lung at moderatelevels, with a weak signal in trachea. Expression was not found in anyother tissues examined. The lung-specific expression of Gene 214implicates it as a gene involved in lung biology and further valicatesas a candidate asthma gene.

3. RT-PCR. RT-PCR was used as an alternate method to Northern blottingto detect mRNAs with low levels of expression. Total RNA from multiplehuman tissues was purchased from Clontech (Palo Alto, Calif.) andgenomic DNA was removed from the total RNA by DNaseI digestion. The“Superscript’ Preamplification System for First strand cDNA synthesis”(Life Technologies, Gaithersburg, Md.) was used according tomanufacturer's specifications with oligo(dT) or random hexamers tosynthesize cDNA from the DNaseI treated total RNA. Gene specific primerswere used to amplify the target cDNAs in a 30 μl PCR reaction containing0.5 μl of first strand cDNA, 1 μl sense primer (10 uM), 1 μl antisenseprimer (10 uM), 3 μl dNTPs (2 mM), 1.2 μl MgCl₂ (25 mM), 3 μl 10×PCRbuffer and 1 unit of Taq Polymerase (Perkin Elmer). The PCR reaction wasinitially denatured at 94° C. for 4 min, then 30 cycles of denaturationat 94° C. for 30 sec, annealing at 58° C. for 1 min and extension at 72°C. for 1 min, followed by a final extension at 72° C. for 7 min. PCRproducts were analyzed on agarose gels.

H. Characteristics and Function of Gene 214

BAC RP11-702C13 (196 Kb) maps to chromosome 12q24 and contains the STSmarker A005Q05 located approximately 165 cM from the telomere of thep-arm of chromosome 12. Gene 214 maps within a 10,318 kb sequencedcontig of the BAC RP11-702C13 (FIG. 2). BLAST analysis against DNA andprotein databases indicated that a portion of Gene 214 was 100%homologous to a nucleic acid sequence known as mucin 8 (MUC8). Northernblot analysis of Gene 214 detected a 4.4 Kb transcript in lung (FIG. 9).The MUC8 fragment is 1.4 Kb in length (Shanker et.al., Am J. Respir.Cell Mol. Biol., 16:232-241 (1997)). Enclosed herein are an additionalfour alternatively transcribed variants. (SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6, SEQ ID NO: 8, and SEQ ID NO:10). The five variants of Gene214/MUC8 contain a putative open reading frames that vary in size, from1167 bp to 1350 bp, and thus encode proteins from 388 to 449 amino acids(SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 9, and SEQ ID NO:11).

Mucin 8 belongs to a growing family of genes that encode mucins.Currently, there are 9 members of this family, MUC1, MUC2, MUC3, MUC4,MUC5, MUC5B, MUC6 and MUC7, and the fragment MUC8. All but MUC3 and MUC6are expressed in the upper and/or lower respiratory tract (Zuhdi Alimamet al., Am. J. Respir. Cell Mol. Biol., 22:253-260 [2000]). All themucins share a common characteristic: tandem repeated amino acidsequences within the protein core. These repeats are rich in serine,threonine and proline and are heavily glycosylated via O-glycosidicbonds. The tandem repeat units vary in length from as few as 8 to asmany as 169 amino acids and are always flanked by non-repeat regions.The MUC8 core protein is unique among the mucins, in that it possesses adegenerate 41 bp tandem repeat that encodes 2 types of consensus peptiderepeats; three 41 bp repeats encode one peptide sequence while a 2 bpdeletion in the perfect 4 lbp repeat disrupts the tandem and generates asecond smaller repetitive portion of the protein (Shanker et.al.,Biochem. J., 300:295-298 (1994).

The respiratory epithelium is protected by a viscoelastic gel, mucus,that is normally produced at low levels. In a healthy individual, 10 mlsof sputum are transported to the larynx and swallowed. In asthmaticindividuals, mucus production is increased. This causes airwayobstruction due to the sputum being very tenacious, and hence formingviscid plugs that can be difficult to expectorate. The overproduction ofmucus in asthmatics has been attributed to the increased numbers ofgoblets cells, goblet cell hyperplasia (GCH), and enlargement of thesub-mucosal glands. GCH is presumed to be due to a combination of mucusgland stimulation by neural stimuli and inflammatory mediators. In situhybridization revealed that multiple airway mucin genes account for thetotal mucin secretion derived from the airway epithelia. Further,immunohistochemical staining of tracheobronchial epithelium withpolyclonal antibodies raised to MUC8, indicated that the protein wasprimarily localized to sub-mucosal glands (Shanker et.al., Am J. Respir.Cell Mol. Biol., 16:232-241 [1997]). Therefore it is likely that therelationship and functional role of Gene 214/MUC8 are involved in thepathophysiology of asthma and other respiratory diseases.

I. Mutation Analysis

In order to conduct mutation analysis, the genomic structure for Gene214 was identified. The precise intron-exon junctions were determinedbased on the consensus sequences at splice junctions. The exonprediction programs MZEF (Zhang, Proc. Natl. Acad. Sci., 94:565-568(1997); and GenScan (Burge and Karlin, J. Mol. Biol., 268:78-94) werealso utilized to help identify the exons.

A combination of fluorescent single stranded confirmation (SSCP)analysis (ABI) and DNA sequencing was utilized to precisely identify anddetermine the nature of the variant at the nucleotide level. SSCPanalysis was used to screen individual DNA for variants. Briefly,polymerase chain reaction (PCR) was used to generate templates fromunrelated asthmatic individuals that showed increased sharing for the12q23-qter chromosomal region and contributed towards linkage.Non-asthmatic individuals were used as controls. Enzymatic amplificationof genes within the asthma region on 12q23-qter was accomplished usingPCR with oligonucleotides flanking each exon as well as the putative 5′regulatory elements of each gene. The primers were chosen to amplifyeach exon as well as 15 or more base pairs within each intron on eitherside of the splice site. The forward and the reverse primers had twodifferent dye colors to allow analysis of each strand and confirmvariants independently. Standard PCR assay was utilized for each exonprimer pair following optimization. Buffer and cycling conditions werespecific to each primer set. The products were denatured using aformamide dye and electrophoresed on non-denaturing acrylamide gels withvarying concentrations of glycerol (at least two different glycerolconcentrations).

Primers utilized in fluorescent SSCP experiments to screen coding andnon-coding regions of Gene 214 for polymorphisms are provided in Table3. Column one lists the gene targeted for mutation analysis. Column twolists the specific exon analyzed. Column three provides the GTC assignedprimer name. Columns four and five list the forward primer sequence andreverse primer sequence, respectively.

TABLE 3 Forward Reverse Gene Exon SSCP Assay Primer Primer 214 A196_214_A_F_197_214_A_R GCCCTTAGGGAGAGCAGC CCACATCGTGCCTTTGTGTA(SEQ ID NO: 24) (SEQ ID NO: 25) 214 B 192_214_B_F_193_214_B_RCACTGTGTTAAAACGCCTGG GTTGGGATTACAGGCACGAG (SEQ ID NO: 26)(SEQ ID NO: 27) 214 B 194_214_B_F_195_214_B_R CAGAAGCAACCCACATGACCACTACAGGTTTGCACCACCA (SEQ ID NO: 28) (SEQ ID NO: 29) 214 C626_214_C_F_627_214_C_R ATGCTCTCCTGATGGCTCCT AGGGAATGCAGGTGCAAAG(SEQ ID NO: 30) (SEQ ID NO: 31) 214 C 628_214_C_F_629_214_C_RACTCGGGAAAGGAAGGCTCT CATACCTTGAGTGCACACCG (SEQ ID NO: 32)(SEQ ID NO: 33)

Primers utilized in DNA sequencing for purposes of confirmingpolymorphisms detected using fluorescent SSCP are provided in Table 4.Column one lists the specific exon sequenced. Column two provides theGTC assigned forward primer name and column three lists the forwardprimer sequence. Columns four and five lists the GTC assigned reverseprimer name and the corresponding reverse primer sequence, respectively.

TABLE 4 Forward Forward Reverse Reverse Gene Exon Primer Sequence PrimerSequence 214 B MDSeq_15_214_B_F GACAGTCTGCTCCACATCCA MDSeq_15_214_B_RTGGAGATGAAGTCTTGCTCTTG (SEQ ID NO: 34) (SEQ ID NO: 35) 214 CMDSeq_110_214_C_F ATATGTTTGCTGGCTTTGGG MDSeq_110_214_C_RCCCAGGCTGTGTGTCCTCTA (SEQ ID NO: 36) (SEQ ID NO: 37)

Single nucleotide polymorphisms (SNPs) that were identified in Gene 214are provided in Table 5. Column one contains the exon or intron in whichthe SNP was detected. Column two provides a reference sequence in whichthe SNP appears underlined. Column three lists the base change of theSNP. Column four details the location of the SNP as intronic or exonic.Column five describes the SNP location of the genomic BAC sequence ofSEQ ID NO:1. The SNPs are also described in FIGS. 8A-8B).

TABLE 5 Reference Intron/ Exon Sequence PMP Exon Location BACTACAGGTTTGCACCACC A A > G Intron 6674 TGTCCTGCTAATTTTTTTTT (SEQ ID(SEQ ID NO: 46) NO: 47) B TGTGCACTCTTGGGCATA C G C > T 3′UTR 6976CCTAGGAGTGGAACTGCTG (SEQ ID (SEQ ID NO: 48) NO: 49) CGGGCTCTGCGCCACCTCAAC C > T Intron 3161 C CAGGCGTTTGTTCCGCAG (SEQ ID(SEQ ID NO: 50) NO: 51)

FIGS. 8A-8B illustrate the five different transcripts of Gene 214 andshow the genomic structure of the gene. The exons are shown to scale andthe SNPs are identified by their location along the genomic BAC DNA (SEQID NO:1).

J. Restriction Fragment Length Polymorphism (RFLP Assay) and AlleleSpecific Oligonucleotide Analysis (ASO Assay)

To identify other individuals with the polymorphisms listed in Table 5,RFLP assay and ASA were performed.

1. RFLP Assay. The amplicon, containing the polymorphism, was PCRamplified using primers that were used to generate a fragment forsequencing (sequencing primers) or SSCP (SSCP primers). The appropriatepopulation of individuals was PCR amplified in 96 well microtitreplates.

Enzymes were purchased from New England Biolabs (NEB). The restrictioncocktail containing the appropriate enzyme for the particularpolymorphism is added to the PCR product. The reaction is incubated atthe appropriate temperature according to the manufacturer'srecommendations (NEB) for two to three hours, followed by a 4° C.incubation. After digestion, the reactions were size fractionated usingthe appropriate agarose gel depending on the assay specifications (2.5%,3%, or metaphor). Gels are electrophoresed in 1×TBE Buffer at 170 Voltsfor approximately two hours.

The gel is illuminated using ultraviolet light and the image is saved asa Kodak 1D file. Using the Kodak 1D image analysis software, the imagesare scored and the data is exported to EXCEL.

2. ASO assay. The amplicon, containing the polymorphism, was PCRamplified using primers that were used to generate a fragment forsequencing (sequencing primers) or SSCP (SSCP primers). The appropriatepopulation of individuals was PCR amplified in 96 well microtitre platesand re-arrayed into 384 well microtitre plates using a Tecan GenesisRSP200. The amplified products were loaded onto 2% agarose gels and sizefractionated at 150V for 5 minutes. The DNA was transferred from the gelto Hybond N+ nylon membrane (Amersham-Pharmacia) using a Vacuum blotter(Bio-Rad). The filter containing the blotted PCR products wastransferred to a dish containing 300 mls of pre-hybridization solution(5×SSPE {pH7.4}, 2% SDS, 5×Denhardts). The filter was left in thepre-hybridization solution at 40° C. for >1 hour. Afterpre-hybridization, 10 mls of the pre-hybridization solution and thefilter were transferred to a washed glass bottle. The allele specificoligonucleotides (ASO) were designed with the polymorphism in themiddle. The size of the oligonucleotide was dependent upon the GCcontent of the sequence around the polymorphism. Those ASOs that had a Gor C polymorphism were designed so that the Tm was between 54-56° C. andthose that had an A or T variance were designed so that the Tm wasbetween 60-64° C. All oligonucleotides were phosphate free at the 5′ endand purchased from Gibco BRL. For each polymorphism 2 ASOs weredesigned: one for each variant.

The two ASOs that represented the polymorphism were resuspended at aconcentration of 1 μg/μl and separately end-labeled with γ-ATP³²(6000Ci/mmol) (NEN) using T4 polynucleotide kinase according tomanufacturer recommendations (NEB). The end-labeled products wereremoved from the unincorporated γ-ATP³² by passing the reactions throughSephadex G-25 columns according to manufacturers recommendation(Amersham-Pharmacia). The entire end-labeled product of one ASO wasadded to the bottle containing the appropriate filter and 10 mls ofhybridization solution. The hybridization reaction was placed in arotisserie oven (Hybaid) and left at 40° C. for a minimum of 4 hours.The other ASO was stored at −20° C.

After the prerequisite hybridization time had elapsed, the filter wasremoved from the bottle and transferred to 1 liter of wash solution(0.1×SSPE {pH7.4}, 0.1% SDS) pre-warmed to 45° C. After 15 minutes thefilter was transferred to another liter of wash solution (0.1×SSPE{pH7.4}, 0.1% SDS) pre-warmed to 50° C. After 15 minutes the filter waswrapped in Saran, placed in an autoradiograph cassette and an X-ray film(Kodak) placed on top of the filter. Depending on the efficiency of theend-labeling reaction of the ASO and its hybridization to the filter animage would be observed on the film within an hour. After an image hadbeen captured on film for the 50° C. wash, the process was repeated forwash steps at 55° C., 60° C. and 65° C. The image that captured the bestresult was used.

The ASO was removed from the filter by adding 1 liter of boiling stripsolution (0.1×SSPE {pH7.4}, 0.1% SDS). This was repeated two more times.After removing the ASO the filter was pre-hybridized in 300 mls ofpre-hybridization solution (5×SSPE {pH7.4}, 2% SDS, 5×Denhardts) at 40°C. for >1 hour. The second end-labeled ASO corresponding to the othervariant was removed from storage at −20° C. and thawed to roomtemperature. The filter was placed into a glass bottle along with 10 mlsof hybridization solution and the entire end-labeled product of thesecond ASO. The hybridization reaction was placed in a rotisserie oven(Hybaid) and left at 40° C. for a minimum of 4 hours. After thehybridization, the filter was washed at various temperatures and imagescaptured on film as described above.

The two films that best captured the allele specific assay with the twoASOs were converted into digital images by scanning them into AdobePhotoShop. These images were overlaid against each other in GraphicConverter and then scored and stored in FileMaker Pro 4.0.

K. Association Study Analysis

In order to determine whether mutations in candidate genes areresponsible for the asthma phenotype, association studies are performedusing a case-control study design. To avoid issues of populationadmixture which can bias case-control studies, the unaffected controlswere collected in both the US and the UK. A total of three hundredcontrols were collected, 200 in the UK and 100 in the US. Inclusion intothe study required that the control individual was negative for asthma,as determined by self report of never having asthma, has no first degreerelatives with asthma, and was negative for eczema and symptomsindicative of atopy within the past 12 months. Data from an abbreviatedquestionnaire similar to that administered to the affected sib pairfamilies were collected. Results from skin prick tests to 4 commonallergens were also collected. The results of the skin prick test wereused to select a subset of control that were most likely to be asthmaand atopy negative.

A subset of unrelated cases are selected from the affected sib pairfamilies based on the evidence for linkage at the chromosomal locationof interest. One affected sib from families demonstratingidentity-by-decent (IBD) at the appropriate marker loci is selected. Inthe selection criteria, preference is given to families with multipleaffected sibs all of whom are concordant at the marker locus as well asto families where affected and unaffected sibs are discordant.

For each polymorphism, the frequency of the alleles in the control andcase populations is compared using a Fisher exact test. It is expectedthat a mutation increasing susceptibility to the disease would be moreprevalent in the cases than in the controls, while a protective mutationshould be more prevalent in the control group. Similarly, the genotypefrequencies of the SNPs are compared between cases and controls.P-values are computed for both the allele and genotype frequencies. Asmall p-value is indicative of an association between the SNPs and thedisease phenotype. The analysis is repeated for the US and UK populationseparately, to adjust for the possibility of genetic heterogeneity.

1. Association Test with Individual SNPs

Statistical analyses for the two SNPs in Gene 214 are presented in Table8. Column one list the exon containing the SNP of interest. The control(“CNTL”) allele frequency and sample size (“N”) are in columns two andthree. The affected individuals (“CASE”) allele frequency and samplesize (“N”) are listed in columns four and five. The sixth columncontains the significance value level of comparison between the controlallele frequencies and the case allele frequencies. The SNP in Exon Chad allelic frequencies significantly different in the cases versus thecontrols in the US and combined samples. In the Combined and USpopulation, this SNP was more frequent in the cases (4.1% and 10.4%,respectively) than in the control population (0.8% and 1.3%), and thedifferences were statistically significance (p=0.0099 and p=0.0083).This analysis suggests that Gene 214, is, at least partially responsiblefor the asthmatic phenotype in those families linked to the chromosome12 region.

TABLE 8 Frequencies ALLELE EXON CNTL N CASE N P-VALUE Combined sample B17.8% 214 20.3% 111 0.4577 C 0.8% 194 4.1% 97 0.0083 US sample B 15.1%76 16.7% 24 0.8204 C 1.3% 75 10.4% 24 0.0099 UK sample B 19.2% 138 21.3%87 0.6291 C 0.4% 119 2.1% 73 0.1559

II. Preparation of Nucleic Acids, Vectors, Transformations and HostCells

The nucleic acids of this invention can be produced in large quantitiesby replication in a suitable host cell. Natural or synthetic nucleicacid fragments, comprising at least ten contiguous bases coding for adesired peptide or polypeptide can be incorporated into recombinantnucleic acid constructs, usually DNA constructs, capable of introductioninto and replication in a prokaryotic or eukaryotic cell. Usually thenucleic acid constructs will be suitable for replication in aunicellular host, such as yeast or bacteria, but may also be intendedfor introduction to (with and without integration within the genome)cultured mammalian or plant or other eukaryotic cells, cell lines,tissues, or organisms. The purification of nucleic acids produced by themethods of the present invention is described, for example, in Sambrooket al, Molecular Cloning. A Laboratory Manual, 2nd Ed. (Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1989) or Ausubel et al,Current Protocols in Molecular Biology, J. Wiley and Sons, NY (1992).

The nucleic acids of the present invention can also be produced bychemical synthesis, e.g., by the phosphoramidite method described byBeaucage et al, Tetra. Letts., 22:1859-1862 (1981) or the triestermethod according to Matteucci, et al, J. Am. Chem. Soc., 103:3185(1981), and can performed on commercial, automated oligonucleotidesynthesizers. A double-stranded fragment may be obtained from thesingle-stranded product of chemical synthesis either by synthesizing thecomplementary strand and annealing the strands together underappropriate conditions or by adding the complementary strand using DNApolymerase with an appropriate primer sequence.

These nucleic acids can encode full-length variant forms of proteins aswell as the naturally-occurring protein. The variant proteins (whichcould be especially useful for detection and treatment of disorders) canhave the variant amino acid sequences encoded by the polymorphismsdescribed in Table 5, when said polymorphisms are read so as to bein-frame with the full-length coding sequence of which it is acomponent.

Nucleic acid constructs prepared for introduction into a prokaryotic oreukaryotic host will comprise a replication system recognized by thehost, including the intended nucleic acid fragment encoding the selectedprotein or polypeptide, and will preferably also include transcriptionand translational initiation regulatory sequences operably linked to theprotein encoding segment. Expression vectors may include, for example,an origin of replication or autonomously replicating sequence (ARS) andexpression control sequences, a promoter, an enhancer and necessaryprocessing information sites, such as ribosome-binding sites, RNA splicesites, polyadenylation sites, transcriptional terminator sequences, andmRNA stabilizing sequences. Secretion signals are also included, whereappropriate, whether from a native Gene 214 protein or from otherreceptors or from secreted proteins of the same or related species,which allow the protein to cross and/or lodge in cell membranes, andthus attain its functional topology, or be secreted from the cell. Suchvectors may be prepared by means of standard recombinant techniques wellknown in the art and discussed, for example, in Sambrook et al,Molecular Cloning. A Laboratory Manual, 2nd Ed. (Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989) or Ausubel et al, CurrentProtocols in Molecular Biology, J. Wiley and Sons, NY (1992).

An appropriate promoter and other necessary vector sequences will beselected so as to be functional in the host, and will include, whenappropriate, those naturally associated with Gene 214 gene. Examples ofworkable combinations of cell lines and expression vectors are describedin Sambrook et al, Molecular Cloning. A Laboratory Manual, 2nd Ed. (ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) or Ausubel etal, Current Protocols in Molecular Biology, J. Wiley and Sons, NY(1992). Many useful vectors are known in the art and can be obtainedfrom such vendors as Stratagene (supra), New England BioLabs, Beverly,Mass., U.S.A, Promega Biotech, and other biotechnology productsuppliers. Promoters such as the trp, lac and phage promoters, tRNApromoters and glycolytic enzyme promoters may be used in prokaryotichosts. Useful yeast promoters include promoter regions formetallothionein, 3-phosphoglycerate kinase or other glycolytic enzymessuch as enolase or glyceraldehyde-3-phosphate dehydrogenase, enzymesresponsible for maltose and galactose utilization, and others. Vectorsand promoters suitable for use in yeast expression are further describedin EP 73,675A. Appropriate non-native mammalian promoters might includethe early and late promoters from SV40 (Fiers et al, Nature, 273:113(1978)) or promoters derived from murine Moloney leukemia virus, mousetumor virus, avian sarcoma viruses, adenovirus II, bovine papillomavirus or polyoma. In addition, the construct may be joined to anamplifiable gene (e.g., DHFR) so that multiple copies of the gene may bemade. For appropriate enhancer and other expression control sequences,see also Enhancers and Eukaryotic Gene Expression, Cold Spring HarborPress, Cold Spring Harbor, N.Y. (1983). While such expression vectorsmay replicate autonomously, they may also replicate by being insertedinto the genome of the host cell, by methods well known in the art.

Expression and cloning vectors will likely contain a selectable marker,a gene encoding a protein necessary for survival or growth of a hostcell transformed with the vector. The presence of this gene ensuresgrowth of only those host cells which express the inserts. Typicalselection genes encode proteins that a) confer resistance to antibioticsor other toxic substances, e.g. ampicillin, neomycin, methotrexate,etc.; b) complement auxotrophic deficiencies, or c) supply criticalnutrients not available from complex media, e.g., the gene encodingD-alanine racemase for Bacilli. The choice of the proper selectablemarker will depend on the host cell, and appropriate markers fordifferent hosts are well known in the art.

The vectors containing the nucleic acids of interest can be transcribedin vitro, and the resulting RNA introduced into the host cell bywell-known methods, e.g., by injection (see, Kubo et al, FEBS Letts.241:119 (1988)), or the vectors can be introduced directly into hostcells by methods well known in the art, which vary depending on the typeof cellular host, including electroporation; transfection employingcalcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, orother substances; microprojectile bombardment; lipofection; infection(where the vector is an infectious agent, such as a retroviral genome);and other methods. See generally, Sambrook et al., 1989 and Ausubel etal., 1992. The introduction of the nucleic acids into the host cell byany method known in the art, including those described above, will bereferred to herein as “transformation.” The cells into which have beenintroduced nucleic acids described above are meant to also include theprogeny of such cells.

Large quantities of the nucleic acids and proteins of the presentinvention may be prepared by expressing the Gene 214 nucleic acids orportions thereof in vectors or other expression vehicles in compatibleprokaryotic or eukaryotic host cells. The most commonly used prokaryotichosts are strains of Escherichia coli, although other prokaryotes, suchas Bacillus subtilis or Pseudomonas may also be used.

Mammalian or other eukaryotic host cells, such as those of yeast,filamentous fungi, plant, insect, or amphibian or avian species, mayalso be useful for production of the proteins of the present invention.Propagation of mammalian cells in culture is per se well known. See,Jakoby and Pastan (eds.), Cell Culture. Methods in Enzymology, volume58, Academic Press, Inc., Harcourt Brace Jovanovich, N.Y., (1979)).Examples of commonly used mammalian host cell lines are VERO and HeLacells, Chinese hamster ovary (CHO) cells, and WI38, BHK, and COS celllines, although it will be appreciated by the skilled practitioner thatother cell lines may be appropriate, e.g., to provide higher expressiondesirable glycosylation patterns, or other features.

Clones are selected by using markers depending on the mode of the vectorconstruction. The marker may be on the same or a different DNA molecule,preferably the same DNA molecule. In prokaryotic hosts, the transformantmay be selected, e.g., by resistance to ampicillin, tetracycline orother antibiotics. Production of a particular product based ontemperature sensitivity may also serve as an appropriate marker.

Prokaryotic or eukaryotic cells transformed with the nucleic acids ofthe present invention will be useful not only for the production of thenucleic acids and proteins of the present invention, but also, forexample, in studying the characteristics of Gene 214 proteins.

Antisense nucleic acid sequences are useful in preventing or diminishingthe expression of Gene 214 gene, as will be appreciated by one skilledin the art. For example, nucleic acid vectors containing all or afragment Gene 214 gene, complementary sequences of the former, or othersequences from the 12q23-qter region may be placed under the control ofa promoter in an antisense orientation and introduced into a cell. Suchfragments can be 16 or more nucleotides in length. Expression of such anantisense construct within a cell will interfere with Gene 214transcription and/or translation and/or replication.

The probes and primers based on the Gene 214 gene sequences disclosedherein are used to identify homologous Gene 214 gene sequences andproteins in other species. These Gene 214 gene sequences and proteinsare used in the diagnostic/prognostic, therapeutic and drug screeningmethods described herein for the species from which they have beenisolated.

III. Protein Expression and Purification

Expression and purification of the Gene 214 protein of the invention canbe performed essentially as outlined below. To facilitate the cloning,expression and purification of membrane and secreted protein from the12q23-qter, a gene expression system, such as the pET System (Novagen),for cloning and expression of recombinant proteins in E. coli isselected. Also, a DNA sequence encoding a peptide tag, the His-Tap, isfused to the 3′ end of DNA sequences of interest to facilitatepurification of the recombinant protein products. The 3′ end is selectedfor fusion to avoid alteration of any 5′ terminal signal sequence.

Nucleic acids chosen, for example, from the nucleic acids set forth SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, and SEQ ID NO:10, orSEQ ID NO:1 for cloning the genes are prepared by polymerase chainreaction (PCR). Synthetic oligonucleotide primers specific for the 5′and 3′ ends of the nucleotide sequences are designed and purchased fromLife Technologies (Gaithersburg, Md.). All forward primers (specific forthe 5′ end of the sequence) are designed to include an NcoI cloning siteat the 5′ terminus. These primers are designed to permit initiation ofprotein translation at the methionine residue encoded within the NcoIsite followed by a valine residue and the protein encoded by the DNAsequence. All reverse primers (specific for the 3′ end of the sequence)include an EcoRI site at the 5′ terminus to permit cloning of thesequence into the reading frame of the pET-28b. The pET-28b vectorprovides a sequence encoding an additional 20 carboxyl-terminal aminoacids including six histidine residues (at the C-terminus), whichcomprise the histidine affinity tag.

DNA prepared from the 12q23-qter region is used as the source oftemplate DNA for PCR amplification (Ausubel et al, Current Protocols inMolecular Biology, John Wilty & Sons (1994)). To amplify a DNA sequencecontaining the nucleotide sequence, cDNA (50 ng) is introduced into areaction vial containing 2 mM MgCl₂, 1 micromolar syntheticoligonucleotide primers (forward and reverse primers) complementary toand flanking a defined 12q23-qter region, 0.2 mM of each ofdeoxynucleotide triphosphate, dATP, dGTP, dCTP, dTTP and 2.5 units ofheat stable DNA polymerase (Amplitaq, Roche Molecular Systems, Inc.,Branchburg, N.J.) in a final volume of 100 microliters.

Upon completion of thermal cycling reactions, each sample of amplifiedDNA is purified using the Qiaquick Spin PCR purification kit (Qiagen,Gaithersburg, Md.). All amplified DNA samples are subjected to digestionwith the restriction endonucleases, e.g., NcoI and EcoRI (New EnglandBioLabs, Beverly, Mass., U.S.A.) (Ausubel et al, Current Protocols inMolecular Biology, John Wiley & Sons, Inc. (1994)). DNA samples are thensubjected to electrophoresis on 1.0% NuSeive (FMC BioProducts, Rockland,Me.) agarose gels. DNA is visualized by exposure to ethidium bromide andlong wave UV irradiation. DNA contained in slices isolated from theagarose gel are purified using the Bio 101 GeneClean Kit protocol (Bio101, Vista, Calif.).

The pET-28b vector is prepared for cloning by digestion with restrictionendonucleases, e.g., NcoI and EcoRI (New England BioLabs, Beverly,Mass.) (Ausubel et al, Current Protocols in Molecular Biology, JohnWiley & Sons, Inc. (1994)). The pET-28a vector, which encodes thehistidine affinity tag that can be fused to the 5′ end of an insertedgene, is prepared by digestion with appropriate restrictionendonucleases.

Following digestion, DNA inserts are cloned (Ausubel et al, CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc. (1994)) into thepreviously digested pET-28b expression vector. Products of the ligationreaction are then used to transform the BL21 strain of E. coli (Ausubelet al, Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994)) as described below.

Competent bacteria, E. coli strain BL21 or E. coli strain BL21 (DE3),are transformed with recombinant pET expression plasmids carrying thecloned sequence according to standard methods (Ausubel et al, CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc. (1994)).Briefly, 1 microliter of ligation reaction is mixed with 50 microlitersof electrocompetent cells and subjected to a high voltage pulse, afterwhich samples were incubated in 0.45 ml SOC medium (0.5% yeast extract,2.0% tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄ and 20mM glucose) at 37° C. with shaking for 1 hour. Samples are then spreadon LB agar plates containing 25 μg/ml kanamycin sulfate for growthovernight. Transformed colonies of BL21 are then picked and analyzed toevaluate cloned inserts, as described below.

Individual BL21 clones transformed with recombinant pET-28b 12q23-qterregion nucleotide sequences are analyzed by PCR amplification of thecloned inserts using the same forward and reverse primers specific forthe 12q23-qter region sequences that are used in the original PCRamplification cloning reactions. Successful amplification verifies theintegration of the sequence in the expression vector (Ausubel et al,Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994)).

Individual clones of recombinant pET-28b vectors carrying properlycloned 12q23-qter region nucleotide sequences are picked and incubatedin 5 ml of LB broth plus 25 μg/mlkanamycin sulfate overnight. Thefollowing day plasmid DNA is isolated and purified using the Qiagenplasmid purification protocol (Qiagen Inc., Chatsworth, Calif.).

The pET vector can be propagated in any E. coli K-12 strain, e.g.,HMS174, HB101, JM109, DH5 and the like, for purposes of cloning orplasmid preparation. Hosts for expression include E. coli strainscontaining a chromosomal copy of the gene for T7 RNA polymerase. Thesehosts are lysogens of bacteriophage DE3, a lambda derivative thatcarries the lad gene, the lacUV5 promoter and the gene for T7 RNApolymerase. T7 RNA polymerase is induced by addition ofisopropyl-β-D-thiogalactoside (IPTG), and the T7 RNA polymerasetranscribes any target plasmid containing a functional T7 promoter, suchas pET-28b, carrying its gene of interest. Strains include, for example,BL21(DE3) (Studier et al, Meth. Enzymol., 185:60-89 (1990)).

To express the recombinant sequence, 50 ng of plasmid DNA are isolatedas described above to transform competent BL21(DE3) bacteria asdescribed above (provided by Novagen as part of the pET expression kit).The lacZ gene (β-galactosidase) is expressed in the pET-System asdescribed for the 12q23-qter region recombinant constructions.Transformed cells were cultured in SOC medium for 1 hour, and theculture is then plated on LB plates containing 25 μg/mlkanamycinsulfate. The following day, the bacterial colonies are pooled and grownin LB medium containing kanamycin sulfate (25 μg/ml) to an opticaldensity at 600 nM of 0.5 to 1.0 O.D. units, at which point 1 mM IPTG wasadded to the culture for 3 hours to induce gene expression of the12q23-qter region recombinant DNA constructions.

After induction of gene expression with IPTG, bacteria are collected bycentrifugation in a Sorvall RC-3B centrifuge at 3500×g for 15 minutes at4° C. Pellets are resuspended in 50 ml of cold mM Tris-HCl, pH 8.0, 0.1M NaCl and 0.1 mM EDTA (STE buffer). Cells are then centrifuged at2000×g for 20 minutes at 4° C. Wet pellets are weighed and frozen at−80° C. until ready for protein purification.

A variety of methodologies known in the art can be used to purify theisolated proteins (Coligan et al, Current Protocols in Protein Science,John Wiley & Sons (1995)). For example, the frozen cells can be thawed,resuspended in buffer and ruptured by several passages through a smallvolume microfluidizer (Model M-110S, Microfluidics International Corp.,Newton, Mass.). The resultant homogenate is centrifuged to yield a clearsupernatant (crude extract) and, following filtration, the crude extractis fractioned over columns. Fractions are monitored by absorbance atOD₂₈₀ nm and peak fractions may be analyzed by SDS-PAGE.

The concentrations of purified protein preparations are quantifiedspectrophotometrically using absorbance coefficients calculated fromamino acid content (Perkins, Eur. J. Biochem., 157:169-180 (1986)).Protein concentrations are also measured by the method of Bradford,Anal. Biochem., 72:248-254 (1976) and Lowry et al, J. Biol. Chem.,193:265-275 (1951) using bovine serum albumin as a standard.

SDS-polyacrylamide gels of various concentrations are purchased fromBioRad (Hercules, Calif.), and stained with Coomassie blue. Molecularweight markers may include rabbit skeletal muscle myosin (200 kDa), E.coli β-galactosidase (116 kDa), rabbit muscle phosphorylase B (97.4kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), bovinecarbonic anyhdrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), eggwhite lysozyme (14.4 kDa) and bovine aprotinin (6.5 kDa).

Proteins can also be isolated by other conventional means of proteinbiochemistry and purification to obtain a substantially pure product,i.e., 80, 95, or 99% free of cell component contaminants, as describedin Jacoby, Methods in Enzymology, Vol. 104, Academic Press, New York(1984); Scoopes, Protein Purification, Principles and Practice, 2^(nd)Ed., Springer-Verlag, New York (1987); and Deutscher (ed.), Guide toProtein Purification, Methods in Enzymology, Vol. 182 (1990). If theprotein is secreted, it can be isolated from the supernatant in whichthe host cell is grown; otherwise, it can be isolated from a lysate ofthe host cells.

Once a sufficient quantity of the desired protein has been obtained, itmay be used for various purposes. One use of the protein or polypeptideis the production of antibodies specific for binding. These antibodiesmay be either polyclonal or monoclonal, and may be produced by in vitroor in vivo techniques well known in the art. Monoclonal antibodies toepitopes of any of the peptides identified and isolated as described canbe prepared from murine hybridomas (Kohler, Nature, 256:495 (1975)). Insummary, a mouse is inoculated with a few micrograms of protein over aperiod of two weeks. The mouse is then sacrificed. The cells thatproduce antibodies are then removed from the mouse's spleen. The spleencells are then fused with polyethylene glycol with mouse myeloma cells.The successfully fused cells are diluted in a microtiter plate andgrowth of the culture is continued. The amount of antibody per well ismeasured by immunoassay methods such as ELISA (Engvall, Meth. Enzymol.,70:419 (1980)). Clones producing antibody can be expanded and furtherpropagated to produce protein antibodies. Other suitable techniquesinvolve in vitro exposure of lymphocytes to the antigenic polypeptides,or alternatively, to selection of libraries of antibodies in phage orsimilar vectors. See Huse et al, Science, 246:1275-1281 (1989). Foradditional information on antibody production see Davis et al, BasicMethods in Molecular Biology, Elsevier, N.Y., Section 21-2 (1989). Suchantibodies are particularly useful in diagnostic assays for detection ofvariant protein forms, or as an active ingredient in a pharmaceuticalcomposition.

III. Transformed Hosts, Development of Pharmaceuticals and ResearchTools

Cells and animals that carry Gene 214 can be used as model systems tostudy and test for substances that have potential as therapeutic agents.The cells are typically cultured mesenchymal stem cells. These may beisolated from individuals with somatic or germline Gene 214.Alternatively, the cell line can be engineered to carry the Gene 214, asdescribed above. After a test substance is applied to the cells, thetransformed phenotype of the cell is determined. Any trait oftransformed cells can be assessed, including respiratory diseasesincluding asthma, atopy, and response to application of putativetherapeutic agents.

IV. Diagnostic Applications

As discussed herein, chromosomal region 12q23-qter has been geneticallylinked to a variety of diseases and disorders. This invention providesnucleic acids and SNPs which can be useful in diagnosing individualswith chromosomal abnormalities linked to these diseases.

Antibody-based diagnostic methods: The invention provides methods fordetecting disease-associated antigenic components in a biologicalsample, which methods comprise the steps of: (i) contacting a samplesuspected to contain a disease-associated antigenic component with anantibody specific for an disease-associated antigen, extracellular orintracellular, under conditions in which a stable antigen-antibodycomplex can form between the antibody and disease-associated antigeniccomponents in the sample; and (ii) detecting any antigen-antibodycomplex formed in step (i) using any suitable means known in the art,wherein the detection of a complex indicates the presence ofdisease-associated antigenic components in the sample. It will beunderstood that assays that utilize antibodies directed againstsequences previously unidentified, or previously unidentified as beingdisease-associated, which sequences are disclosed herein, are within thescope of the invention.

Many immunoassay formats are known in the art, and the particular formatused is determined by the desired application. An immunoassay can use,for example, a monoclonal antibody directed against a singledisease-associated epitope, a combination of monoclonal antibodiesdirected against different epitopes of a single disease-associatedantigenic component, monoclonal antibodies directed towards epitopes ofdifferent disease-associated antigens, polyclonal antibodies directedtowards the same disease-associated antigen, or polyclonal antibodiesdirected towards different disease-associated antigens. Protocols canalso, for example, use solid supports, or may involveimmunoprecipitation.

Typically, immunoassays use either a labeled antibody or a labeledantigenic component (e.g., that competes with the antigen in the samplefor binding to the antibody). Suitable labels include without limitationenzyme-based, fluorescent, chemiluminescent, radioactive, or dyemolecules. Assays that amplify the signals from the probe are alsoknown, such as, for example, those that utilize biotin and avidin, andenzyme-labeled immunoassays, such as ELISA assays.

Kits suitable for antibody-based diagnostic applications typicallyinclude one or more of the following components:

-   -   (i) Antibodies: The antibodies can be pre-labeled;        alternatively, the antibody may be unlabeled and the ingredients        for labeling can be included in the kit in separate containers,        or a secondary, labeled antibody is provided; and    -   (ii) Reaction components: The kit can also contain other        suitably packaged reagents and materials needed for the        particular immunoassay protocol, including solid-phase matrices,        if applicable, and standards.

The kits referred to above can include instructions for conducting thetest. Furthermore, in preferred embodiments, the diagnostic kits areadaptable to high-throughput and/or automated operation.

Nucleic-acid-based diagnostic methods: The invention provides methodsfor detecting disease-associated nucleic acids in a sample, such as in abiological sample, which methods comprise the steps of: (i) contacting asample suspected to contain a disease-associated nucleic acid with oneor more disease-associated nucleic acid probes under conditions in whichhybrids can form between any of the probes and disease-associatednucleic acid in the sample; and (ii) detecting any hybrids formed instep (i) using any suitable means known in the art, wherein thedetection of hybrids indicates the presence of the disease-associatednucleic acid in the sample. To detect disease-associated nucleic acidspresent in low levels in biological samples, it may be necessary toamplify the disease-associated sequences or the hybridization signal aspart of the diagnostic assay. Techniques for amplification are known tothose of skill in the art.

Disease-associated nucleic acids useful as probes in diagnostic methodsinclude oligonucleotides at least about 15 nucleotides in length,preferably at least about 20 nucleotides in length, and most preferablyat least about 25-55 nucleotides in length, that hybridize specificallywith one or more disease-associated nucleic acids.

A sample to be analyzed, such as, for example, a tissue sample, may becontacted directly with the nucleic acid probes. Alternatively, thesample may be treated to extract the nucleic acids contained therein. Itwill be understood that the particular method used to extract DNA willdepend on the nature of the biological sample. The resulting nucleicacid from the sample may be subjected to gel electrophoresis or othersize separation techniques, or, the nucleic acid sample may beimmobilized on an appropriate solid matrix without size separation.

Kits suitable for nucleic acid-based diagnostic applications typicallyinclude the following components:

-   -   (i) Probe DNA: The probe DNA may be prelabeled; alternatively,        the probe DNA may be unlabeled and the ingredients for labeling        may be included in the kit in separate containers; and    -   (ii) Hybridization reagents: The kit may also contain other        suitably packaged reagents and materials necessary or desirable        for the particular hybridization protocol, including solid-phase        matrices, if applicable, and standards.

In cases where a disease condition is suspected to involve an alterationof the disease gene, specific oligonucleotides may be constructed andused to assess the level of disease mRNA in cells affected or othertissue affected by the disease.

For example, to test whether a person has a disease gene, polymerasechain reaction can be used. In order to identify an individual whopossesses the disease gene or the wild type copy, two oligonucleotidesare synthesized by standard methods or are obtained from a commercialsupplier of custom-made oligonucleotides. The length and basecomposition are determined by standard criteria using the Oligo 4.0primer Picking program (Wojchich Rychlik, 1992). One of theoligonucleotides is designed so that it will hybridize only to thedisease gene DNA under the PCR conditions used. The otheroligonucleotide is designed to hybridize to a segment of genomic DNA,wild type or non disease gene such that amplification of DNA using theseoligonucleotide primers produces a conveniently identified DNA fragment.Tissue samples may be obtained from hair follicles, whole blood, or thebuccal cavity. The DNA fragment generated by this procedure is sequencedby standard techniques.

Other amplification techniques besides PCR may be used as alternatives,such as ligation-mediated PCR or techniques involving Q-beta replicase(Cahill et al, Clin. Chem., 37(9):1482-5 (1991)). Products ofamplification can be detected by agarose gel electrophoresis,quantitative hybridization, or equivalent techniques for nucleic aciddetection known to one skilled in the art of molecular biology (Sambrooket al, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring, N.Y. (1989)). Other alterations in the diseasegene may be diagnosed by the same type of amplification-detectionprocedures, by using oligonucleotides designed to identify thosealterations.

V. Genomic Screening

The use of polymorphic genetic markers linked to the Gene 214 gene isvery useful in predicting susceptibility to the diseases geneticallinked to 12q23-qter. Similarly, as provided in Table 5 theidentification of polymorphic genetic markers within the Gene 214 genewill allow the identification of specific allelic variants that are inlinkage disequilibrium with other genetic lesions that affect one of thedisease states discussed herein including respiratory disorders andobesity. SSCP allows the identification of polymorphisms within thegenomic and coding region of the disclosed gene. Table 3 providesprimers which one skilled in the art could identify exons which containSNP's. Table 4 provides primers to identify the sequence change. Thisinformation can assist one skilled in the art to identify additionalSNP's for use in genomic screening.

This method has been used successfully by others skilled in the art(e.g., Sheffield et al, Genet., 4:1837-1844 (1995); LeBlanc-Straceski etal, Genomics, 19:341-9 (1994); Chen et al, Genomics, 25:1-8 (1995)). Useof these reagents with populations or individuals will predict theirrisk for diseases or disorders described herein, especially respiratorydisorders and obesity.

VI. Treatment of Disorders

Thus, the present invention provides methods of screening for drugscomprising contacting such an agent with a novel protein of thisinvention or fragment thereof and assaying (i) for the presence of acomplex between the agent and the protein or fragment, or (ii) for thepresence of a complex between the protein or fragment and a ligand, bymethods well known in the art. In such competitive binding assays thenovel protein or fragment is typically labeled. Free protein or fragmentis separated from that present in a protein:protein complex, and theamount of free (i.e., uncomplexed) label is a measure of the binding ofthe agent being tested to the novel protein or its interference withprotein ligand binding, respectively.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of specifically bindingthe Gene 214 proteincompete with a test compound for binding to the Gene214 protein or fragments thereof. In this manner, the antibodies can beused to detect the presence of any peptide which shares one or moreantigenic determinants of a Gene 214 protein.

The goal of rational drug design is to produce structural analogs ofbiologically active proteins of interest or of small molecules withwhich they interact (e.g., agonists, antagonists, inhibitors) in orderto fashion drugs which are, for example, more active or stable forms ofthe protein, or which, e.g., enhance or interfere with the function of aprotein in vivo. See, e.g., Hodgson, Bio/Technology, 9:19-21 (1991). Inone approach, one first determines the three-dimensional structure of aprotein of interest or, for example, of the Gene 214 receptor or ligandcomplex, by x-ray crystallography, by computer modeling or mosttypically, by a combination of approaches. Less often, usefulinformation regarding the structure of a protein may be gained bymodeling based on the structure of homologous proteins. An example ofrational drug design is the development of HIV protease inhibitors(Erickson et al, Science, 249:527-533 (1990)). In addition, peptides(e.g., Gene 214 protein) are analyzed by an alanine scan (Wells, Methodsin Enzymol., 202:390-411 (1991)). In this technique, an amino acidresidue is replaced by Ala, and its effect on the peptide's activity isdetermined. Each of the amino acid residues of the peptide is analyzedin this manner to determine the important regions of the peptide.

It is also possible to isolate a target-specific antibody, selected by afunctional assay, and then to solve its crystal structure. In principle,this approach yields a pharmacore upon which subsequent drug design canbe based. It is possible to bypass protein crystallography altogether bygenerating anti-idiotypic antibodies (anti-ids) to a functional,pharmacologically active antibody. As a mirror image of a minor image,the binding site of the anti-ids would be expected to be an analog ofthe original receptor. The anti-id could then be used to identify andisolate peptides from banks of chemically or biologically produced banksof peptides. Selected peptides would then act as the pharmacore.

Thus, one may design drugs which have, e.g., improved Gene 214proteinactivity or stability or which act as inhibitors, agonists,antagonists, etc. of Gene 214 proteinactivity. By virtue of theavailability of cloned Gene 214 gene sequences, sufficient amounts ofthe Gene 214 protein may be made available to perform such analyticalstudies as x-ray crystallography. In addition, the knowledge of the Gene214 protein sequence will guide those employing computer modelingtechniques in place of, or in addition to x-ray crystallography.

Cells and animals that carry the Gene 214 gene or an analog thereof canbe used as model systems to study and test for substances that havepotential as therapeutic agents. After a test substance is applied tothe cells, the transformed phenotype of the cell is determined.

The therapeutic agents and compositions of the present invention areuseful for preventing or treating respiratory disease. Pharmaceuticalformulations suitable for therapy comprise the active agent inconjunction with one or more biologically acceptable carriers. Suitablebiologically acceptable carriers include, but are not limited to,phosphate-buffered saline, saline, deionized water, or the like.Preferred biologically acceptable carriers are physiologically orpharmaceutically acceptable carriers.

The compositions include an effective amount of active agent. Effectiveamounts are those quantities of the active agents of the presentinvention that afford prophyladic protection against a respiratorydisease, or which result in amelioration or cure of an existingrespiratory disease. Prophylactic methods incorporate a prophylacticallyeffective amount of an active agent or composition. A prophylacticallyeffective amount is an amount effective to prevent disease. Treatmentmethods incorporate a therapeutically effective amount of an activeagent or composition. A therapeutically effective amount is an amountsufficient to ameliorate or eliminate the symptoms of disease. Theeffective amount will depend upon the agent, the severity of disease andthe nature of the disease, and the particular host. The amount can bedetermined by experimentation known in the art, such as by establishinga matrix of dosage amounts and frequencies of dosage administration andcomparing a group of experimental units or subjects to each point in thematrix. The prophylactically and/or therapeutically effective amountscan be administered in one administration or over repeatedadministrations. Therapeutic administration can be followed byprophylactic administration, once initial clinical symptoms of diseasehave been resolved.

The agents and compositions can be administered topically orsystemically. Systemic administration includes both oral and parentalroutes. Parental routes include, without limitation, subcutaneous,intramuscular, intraperitoneal, intravenous, transdermal, and intranasaladministration.

VII. Gene Therapy

In recent years, significant technological advances have been made inthe area of gene therapy for both genetic and acquired diseases. (Kay etal, Proc. Natl. Acad. Sci. USA, 94:12744-12746 (1997)) Gene therapy canbe defined as the deliberate transfer of DNA for therapeutic purposes.Improvement in gene transfer methods has allowed for development of genetherapy protocols for the treatment of diverse types of diseases. Genetherapy has also taken advantage of recent advances in theidentification of new therapeutic genes, improvement in both viral andnonviral gene delivery systems, better understanding of gene regulation,and improvement in cell isolation and transplantation. Gene therapywould be carried out according to generally accepted methods asdescribed by, for example, Friedman, Therapy for Genetic Diseases,Friedman, Ed., Oxford University Press, pages 105-121 (1991).

Vectors for introduction of genes both for recombination and forextrachromosomal maintenance are known in the art, and any suitablevector may be used. Methods for introducing DNA into cells such aselectroporation, calcium phosphate co-precipitation, and viraltransduction are known in the art, and the choice of method is withinthe competence of one skilled in the art (Robbins, Ed., Gene TherapyProtocols, Human Press, NJ (1997)). Cells transformed with a Gene 214gene can be used as model systems to study chromosome 12 disorders andto identify drug treatments for the treatment of such disorders.

Gene transfer systems known in the art may be useful in the practice ofthe gene therapy methods of the present invention. These include viraland nonviral transfer methods. A number of viruses have been used asgene transfer vectors, including polyoma, i.e., SV40 (Madzak et al, J.Gen. Virol., 73:1533-1536 (1992)), adenovirus (Berkner, Curr. Top.Microbiol. Immunol., 158:39-61 (1992); Berkner et al, Bio Techniques,6:616-629 (1988); Gorziglia et al, J. Virol., 66:4407-4412 (1992);Quantin et al, Proc. Natl. Acad. Sci. USA, 89:2581-2584 (1992);Rosenfeld et al, Cell, 68:143-155 (1992); Wilkinson et al, Nucl. AcidsRes., 20:2233-2239 (1992); Stratford-Perricaudet et al, Hum. Gene Ther.,1:241-256 (1990)), vaccinia virus (Mackett et al, Biotechnology,24:495-499 (1992)), adeno-associated virus (Muzyczka, Curr. Top.Microbiol. Immunol., 158:91-123 (1992); Ohi et al, Gene, 89:279-282(1990)), herpes viruses including HSV and EBV (Margolskee, Curr. Top.Microbiol. Immunol., 158:67-90 (1992); Johnson et al, J. Virol.,66:2952-2965 (1992); Fink et al, Hum. Gene Ther., 3:11-19 (1992);Breakfield et al, Mol. Neurobiol., 1:337-371 (1987;) Fresse et al,Biochem. Pharmacol., 40:2189-2199 (1990)), and retroviruses of avian(Brandyopadhyay et al, Mol. Cell. Biol., 4:749-754 (1984); Petropouploset al, J. Virol., 66:3391-3397 (1992)), murine (Miller, Curr. Top.Microbiol. Immunol., 158:1-24 (1992); Miller et al, Mol. Cell. Biol.,5:431-437 (1985); Sorge et al, Mol. Cell. Biol., 4:1730-1737 (1984);Mann et al, J. Virol., 54:401-407 (1985)), and human origin (Page et al,J. Virol., 64:5370-5276 (1990); Buchschalcher et al, J. Virol.,66:2731-2739 (1992)). Most human gene therapy protocols have been basedon disabled murine retroviruses.

Nonviral gene transfer methods known in the art include chemicaltechniques such as calcium phosphate coprecipitation (Graham et al,Virology, 52:456-467 (1973); Pellicer et al, Science, 209:1414-1422(1980)), mechanical techniques, for example microinjection (Anderson etal, Proc. Natl. Acad. Sci. USA, 77:5399-5403 (1980); Gordon et al, Proc.Natl. Acad. Sci. USA, 77:7380-7384 (1980); Brinster et al, Cell,27:223-231 (1981); Constantini et al, Nature, 294:92-94 (1981)),membrane fusion-mediated transfer via liposomes (Feigner et al, Proc.Natl. Acad. Sci. USA, 84:7413-7417 (1987); Wang et al, Biochemistry,28:9508-9514 (1989); Kaneda et al, J. Biol. Chem., 264:12126-12129(1989); Stewart et al, Hum. Gene Ther., 3:267-275 (1992); Nabel et al,Science, 249:1285-1288 (1990); Lim et al, Circulation, 83:2007-2011(1992)), and direct DNA uptake and receptor-mediated DNA transfer (Wolffet al, Science, 247:1465-1468 (1990); Wu et al, BioTechniques,11:474-485 (1991); Zenke et al, Proc. Natl. Acad. Sci. USA, 87:3655-3659(1990); Wu et al, J. Biol. Chem., 264:16985-16987 (1989); Wolff et al,BioTechniques, 11:474-485 (1991); Wagner et al, 1990; Wagner et al,Proc. Natl. Acad. Sci. USA, 88:4255-4259 (1991); Cotten et al, Proc.Natl. Acad. Sci. USA, 87:4033-4037 (1990); Curiel et al, Proc. Natl.Acad. Sci. USA, 88:8850-8854 (1991); Curiel et al, Hum. Gene Ther.,3:147-154 (1991)).

In an approach which combines biological and physical gene transfermethods, plasmid DNA of any size is combined with apolylysine-conjugated antibody specific to the adenovirus hexon protein,and the resulting complex is bound to an adenovirus vector. Thetrimolecular complex is then used to infect cells. The adenovirus vectorpermits efficient binding, internalization, and degradation of theendosome before the coupled DNA is damaged.

Liposome/DNA complexes have been shown to be capable of mediating directin vivo gene transfer. While in standard liposome preparations the genetransfer process is non-specific, localized in vivo uptake andexpression have been reported in tumor deposits, for example, followingdirect in situ administration (Nabel, Hum. Gene Ther., 3:399-410(1992)).

VIII. Transgenic Animals

This invention further relates to nonhuman transgenic animals capable ofexpressing an exogenous or non-naturally occurring variant Gene 214gene. Such a transgenic animal can also have one or more endogenousgenes inactivated or can, instead of expressing an exogenous variantgene, have one or more endogenous analogs inactivated. Any nonhumananimal can be used; however typical animals are rodents, such as mice,rats, or guinea pigs.

Animals for testing therapeutic agents can be selected after treatmentof germline cells or zygotes. Thus, expression of an exogenous Gene 214gene or a variant can be achieved by operably linking the gene to apromoter and optionally an enhancer, and then microinjecting theconstruct into a zygote. See, e.g., Hogan, et al., Manipulating theMouse Embryo, A Laboratory Manual, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. Such treatments include insertion of the exogenousgene and disrupted homologous genes. Alternatively, the gene(s) of theanimals may be disrupted by insertion or deletion mutation of othergenetic alterations using conventional techniques, such as thosedescribed by, for example, Capecchi, Science, 244:1288 (1989); Valancuiset al, Mol. Cell. Biol., 11:1402 (1991); Hasty et al, Nature, 350:243(1991); Shinkai et al, Cell, 68:855 (1992); Mombaerts et al, Cell,68:869 (1992); Philpott et al, Science, 256:1448 (1992); Snouwaert etal, Science, 257:1083 (1992); Donehower et al, Nature, 356:215 (1992).After test substances have been administered to the animals, modulationof the disorder must be assessed. If the test substance reduces theincidence of the disorder, then the test substance is a candidatetherapeutic agent. These animal models provide an extremely importantvehicle for potential therapeutic products.

The disclosure of each of the patents, patent applications andpublications cited in the specification is hereby incorporated byreference herein in its entirety.

Although the invention has been set forth in detail, one skilled in theart will recognize that numerous changes and modifications can be made,and that such changes and modifications may be made without departingfrom the spirit and scope of the invention.

1. An isolated polypeptide encoded by a nucleic acid molecule comprisingthe nucleotide sequence selected from the group consisting of: a) SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10; andb) a nucleotide sequence comprising at least 500 consecutive nucleotidesof SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO. 6, SEQ ID NO. 8, or SEQ ID NO.10.
 2. An isolated polypeptide encoded by a nucleic acid fragmentcomprising at least 500 consecutive nucleotide bases of the nucleotidesequence SEQ ID NO:
 1. 3. The isolated polypeptide of claim 1, whereinthe nucleotide sequence is SEQ ID NO:
 2. 4. The isolated polypeptide ofclaim 1, wherein the nucleotide sequence is SEQ ID NO:
 4. 5. Theisolated polypeptide of claim 1, wherein the nucleotide sequence is SEQID NO:
 6. 6. The isolated polypeptide of claim 1, wherein the nucleotidesequence is SEQ ID NO:
 8. 7. The isolated polypeptide of claim 1,wherein the nucleotide sequence is SEQ ID NO:
 10. 8. The isolatedpolypeptide of claim 1, wherein the nucleotide sequence comprises atleast 500 consecutive nucleotides of SEQ ID NO:
 2. 9. The isolatedpolypeptide of claim 1, wherein the nucleotide sequence comprises atleast 500 consecutive nucleotides of SEQ ID NO:
 4. 10. The isolatedpolypeptide of claim 1, wherein the nucleotide sequence comprises atleast 500 consecutive nucleotides of SEQ ID NO:
 6. 11. The isolatedpolypeptide of claim 1, wherein the nucleotide sequence comprises atleast 500 consecutive nucleotides of SEQ ID NO:
 8. 12. The isolatedpolypeptide of claim 1, wherein the nucleotide sequence comprises atleast 500 consecutive nucleotides of SEQ ID NO: 10.